IL28B gene (e.g. rs12979860) encoding interferon λ3 have recently been associated with both spontaneous clearance and treatment response to pegIFN/RBV in chronic hepatitis C (CHC) patients. The molecular consequences of this genetic variation are unknown. To gain further insight into IL28B function we assessed the association of rs12979860 with expression of protein quantitative traits  generated using open-platform proteomics in serum from patients.Polymorphisms near the IL28B genotype using one-way analysis of variance.41 patients with genotype 1 chronic hepatitis C infection from the Duke Liver Clinic were genotyped for rs12979860. Proteomic profiles were generated by LC-MS/MS analysis following immunodepletion of serum with MARS14 columns and trypsin-digestion. Next, a latent factor model was used to classify peptides into metaproteins based on co-expression and using only those peptides with protein identifications. Metaproteins were then analyzed for association with IL28B treatment response genotype (rs12979860_CC) was significantly associated with lower serum levels of corticosteroid binding globulin , a major transport protein for glucocorticoids and progestins. Moreover, the CBG metaprotein was associated with treatment response (p = 0.0148), but this association was attenuated when both IL28B genotype and CBG were included in the model, suggesting that the CBG association may be independent of treatment response.There were a total of 4,186 peptides in the data set with positive identifications. These were matched with 253 proteins of which 110 had two or more associated, identified peptides. The IL28B polymorphism was associated with serum levels of corticosteroid binding globulin, a major transporter of cortisol, however, CBG does not appear to mediate the association of IL28B with treatment response. Further investigation of this pathway is warranted to determine if it plays a role in other comorbidities of HCV-infection.In this cohort of chronic hepatitis C patients,  IL28B gene, encoding interferon λ3, have been associated with both spontaneous clearance and virologic response to pegylated interferon-alpha and Ribavirin therapy (pegIFN/RBV) among patients with CHC Hepatitis C virus (HCV) infecton is a global health concern with an estimated prevalence of up to 170 million people infected worldwide IL28B SNP association with liver gene expression levels of ISGs and found that hepatic ISGs were significantly lower in HCV patients with the IL28B ‘responder’ genotype compared to patients without this genotype IL28B influences viral clearance.CHC patients who respond to pegIFN/RBV tend to have lower pre-treatment expression levels of interferon stimulated genes (ISGs) in the liver IL28B genotype. Interestingly, the proteomic signature and IL28B genotype demonstrated only partial correlation, suggesting unique pathways to predict response. Serum proteomic data from that investigation were utilized in the current study to explore the correlation of genetic variation with downstream protein expression. This protein quantitative trait locus (pQTL) analysis builds on the success of eQTL analysis, where correlation of genetic variation with downstream mRNA expression has provided insights into the functional consequence of genetic variation. Prior studies employing similar methods have demonstrated the effectiveness of such an approach for identifying genetic variants controlling protein expression in yeast cis-acting (located close to the gene encoding the protein) and trans-acting (located elsewhere in the genome) loci. In the current study we use an unbiased, label-free proteomic platform to characterize serum proteins associated with an IL28B polymorphism that has been strongly associated with HCV clearance.We recently assessed a cohort of CHC patients and reported a proteomic signature of HCV treatment response measured in pre-treatment serum samples that was highly predictive of response to peg-interferon/Ribavirin IL28B CC genotype was observed in close to one-half of our cohort. The study included 41 genotype 1 CHC patients that were predominantly Caucasian (78%), male (61%), treatment responders (63%) with moderate to advanced fibrosis (85%) and a mean age of 47.4 years. The favorable response IL28B genotype after Bonferroni correction for multiple hypothesis testing: the liver protein corticosteroid binding globulin . The rs12979860 ‘C’ (responder) allele was associated with lower levels of CBG metaprotein and explained 46% of the variance in CBG metaprotein (−5).There were a total of 4,186 peptides in the data set with identifications by MS/MS at a 1% false discovery rate. Latent factor modeling grouped the individual peptides into 110 metaproteins. P-values generated from the association analysis of each of the 110 metaproteins and rs12979860 genotype are shown in the quantile-quantile (QQ) plot in aprotein . These rIL28B and SVR is mediated through CBG, we would expect to see an association between CBG and SVR in this cohort as well. Indeed, we found a statistically significant association between the CBG metaprotein expression and SVR (p = 0.0148). We also found a statistically significant association (p = 0.0021) between the IL28B genotype and SVR, as reported previously in patients from the Duke Hepatology Clinic cohort IL28B genotype and CBG in a model to predict SVR, the association with CBG was completely attenuated (p = 0.46) while the IL28B association remained significant (p = 0.0145), suggesting that CBG is probably not mediating the association between IL28B and SVR. These results are summarized in If the previously reported association between IL28B genotype to determine whether all or only some of the peptides contributed to the association observed with the metaprotein  of the CBG peptides generated, as well as individual peptides from three other proteins . As expeaprotein . All eigIL28B gene locus, a strong determinant of treatment-induced HCV clearance. Corticosteroid binding globulin peptides were strongly associated with IL28B genotype, accounting for over a third of the variance of the CBG metaprotein and thus, IL28B may be an important trans-pQTL for serum CBG levels among HCV-infected individuals. We were unable to show that CBG mediates the relationship between IL28B and treatment response, indicating possible pleiotropic effects of this locus.We recently reported the successful application of an unbiased LC-MS/MS platform for proteomic expression signatures associated with HCV treatment response CBG, also known as Transcortin, is a major high-affinity plasma transport protein for glucocorticoids and progestins and is expressed primarily in the liver. Serum cortisol and CBG levels are inversely related IL28B rs12979860-CC responder genotype and advanced liver disease areas associated with lower CBG levels, but IL28B rs12979860-CC is not associated with a lower rate, but rather a more rapid rate of hepatic fibrosis in CHC patients in vitro studies have demonstrated an inhibitory effect of insulin and insulin-like growth factor-1 on CBG mRNA expression in a human hepatoblastoma derived Hep G2 cell-line IL28B rs12979860-C responder allele  to be associated with low serum levels of the CBG metaprotein.Levels of serum CBG may be altered in chronic disease states, but are unlikely to confound our results. CBG levels may be reduced in patients with advanced stage liver disease IL28B and CBG in our study, the results were based on a relatively small number of patients and should thus be considered preliminary. Post-pubertal CBG levels do not vary significantly with age or gender, and there is minimal diurnal variation, thus reducing the potential influence of normal physiological variation on our observations IL28B genotype and corticosteroids.Despite strong statistical support for the observed association between The current study utilized proteomic data generated by LC-MS/MS and analyzed using novel methods to group peptides into ‘metaprotein’ clusters. Thus, the intact proteins were not measured directly, but rather, specific groups of peptides representing the protein were measured. The metaprotein algorithm preferentially, but not exclusively, groups identified peptides according to their assigned “parent” protein. In addition, the algorithm permits exclusion of a peptide from the model if its expression trend does not track with the other peptides, and inclusion of peptides from different proteins or unidentified peptides which have expression in common with the metaprotein. In this way, future work correlating metaproteins with genes identified in pQTL analysis may assist in identification of currently unidentified proteins, or at least functional correlation of these unknown peptides with biological mechanisms.trans-acting pQTL, providing insight into the biological consequences of this variation. We have found the CBG metaprotein and its constitutent peptides to be strongly associated with IL28B genotype, but not mediating the effect of IL28B genotype on treatment response. Our research showcases the power of pQTL analysis for the functional annotation of genetic variants.In summary, the current study used an open-proteomic platform to characterize protein expression associated with a polymorphism strongly associated with HCV clearance and have identified a strong Patient samples were selected from the Duke Hepatology Clinical Research (DHCR) database and were included in a prior validation study of proteomic signatures of virologic response to current IFN-based standard-of-care therapy IL28B gene. We selected the most strongly associated SNP, rs12979860, located upstream of this gene for genotyping in our cohort using the 5′ nuclease assay with allele specific TaqMan probes The genomic region associated with HCV response in Sample preparation and LC-MS/MS analyses on these samples have been described previously Several challenges exist in the analysis of proteomic data generated by LC-MS/MS. Incorrect peptide identifications, sharing of peptides among homologs, and post-translationally modified or processed peptides that may show a biologically relevant and different expression pattern than the proteotypic peptides, can all lead to errors in the quantitative analysis of proteins. In order to correct for these multiple sources of noise, we utilized our metaprotein factor model which groups peptides based on co-expression. We restricted our analysis to only those peptides with protein identifications. Because co-expression across the biological cohort is a central tenant of this modeling approach, peptides are not exclusively tied to their parent protein and therefore individual peptides arising from multiple proteins may potentially comprise a single metaprotein. Metaprotein nomenclature was based on predominance of peptides associated with a known protein. Complete details of how metaprotein expression levels are generated as well as mathematical details regarding the metaprotein factor model are described in IL28B genotype using one-way analysis of variance (ANOVA). IL28B genotypes  were analyzed assuming an additive model, controlling for sex and race. Subsequent analysis was carried out assuming a recessive model for the response allele (C), comparing CC to CT/TT genotypes, consistent with the effect of this locus on treatment response. All statistical analyses have been performed using MATLAB; a commented script Click here for additional data file.Table S1Protein identification frequency in samples from 41 CHC patients. This table lists each of the proteins identified in the data set along with the proteins' full names and the number of different isotope groups from that protein that were identified.(DOC)Click here for additional data file.Materials S1Matlab script.(M)Click here for additional data file.Materials S2Matlab workspace.(MAT)Click here for additional data file.


Compared to individuals with low genetic scores of metal processing gene variants, participants with higher scores had greater effects of particle number on fibrinogen (pinteraction = 0.12), CRP (pinteraction = 0.02), and ICAM-1 (pinteraction = 0.08). This two-stage penalization method is easy to implement and can be used for large-scale genetic applications.Air pollution has been associated with increased systemic inflammation markers. We developed a new pathway analysis approach to investigate whether gene variants within relevant pathways  modified the association between particulate air pollution and fibrinogen, C-reactive protein (CRP), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). Our study population consisted of 822 elderly participants of the Normative Aging Study (1999–2011). To investigate the role of biological mechanisms and to reduce the number of comparisons in the analysis, we created pathway-specific scores using gene variants related to each pathway. To select the most appropriate gene variants, we used the least absolute shrinkage and selection operator (Lasso) to relate independent outcomes representative of each pathway  to gene variants. A high genetic score corresponds to a higher allelic risk profile. We fit mixed-effects models to examine modification by the genetic score of the weekly air pollution association with the outcome. Among participants with higher genetic scores within the oxidative stress pathway, we observed significant associations between particle number and fibrinogen, while we did not find any association among participants with lower scores (p To better understand molecular mechanisms, one can investigate the role of gene variants individually or collectively within a biological pathway. Pathway analysis may be more informative of the underlying biology. Mechanisms by which particulate air pollution is linked to exacerbation of cardiovascular disease (CVD) morbidity and mortality are not fully understood To better understand the molecular mechanisms linking air pollution and CVD, we chose to examine the role gene variants play in modifying the adverse air pollution effects by considering genetic pathway-air pollution interactions This study builds upon previous research by investigating three potential pathways  that may modify the association between air pollution and the outcomes fibrinogen, C-reactive protein, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), which have been related to coronary heart disease and atherosclerosis This study included participants from the Normative Aging Study (NAS), a prospective cohort of aging established in 1963, enrolling men from the Greater Boston area. Participants underwent medical examinations every three to five years. More detailed longitudinal characteristics on the study population can be found elsewhere This study was approved by the Harvard School of Public Health and the Veteran Administration Institution Review Boards (IRB). Participants provided written informed consent to participate in this study, which was approved by the Veteran Administration IRB.3 (0.007–3 µm) with a Condensation Particle Counter , hourly ambient fine particle (PM2.5) concentrations with a Tapered Element Oscillation Microbalance , and hourly PM2.5 black carbon concentrations using an Aethalometer .We measured particulate concentrations at the Harvard supersite located 1 km from the examination site. We measured hourly particle number per cma priori considered only concentrations averaged over the week preceding each participant's examination.The relevant exposure window for gene-air pollution interactions on coagulation, and inflammation is unclear. Results of previous studies suggested an association between air pollution and blood markers of inflammation and coagulation spread over several days and weeks prior to the time of assessment GeneCardsAn exhaustive list of the genes we considered can be found in Tables S1 to S3 in Assays were genotyped using the Sequenom MassArray MALDI-TOF Mass Spectrometer with semi-automated primer design and implementation of the short extension method . The MassARRAY System offers several features including very high sensitivity, capacity to analyze multiple classes of genetic markers, ability to quickly create and modify customized assay panels, flexible sample throughput (from few to thousands), and high quality data with low operating costs. Multiplexed polymerase chain reaction assays were designed using the Sequenom Spectro Designer software .At each visit, we measured concentrations of plasma fibrinogen using MDA Fibriquick , a bovine thrombin reagent . We quantified C-reactive protein levels using an immunoturbidimetric assay on the Hitachi 917 analyzer . We assessed plasma ICAM-1 and VCAM-1 concentrations with an enzyme-linked immunoabsorbent assay method . This technique has been exhaustively tested for superior quality and reproducibility.We related gene variants to each pathway based on their functionality. Because of the large number of gene variants for each gene and within each pathway, we chose to penalize them using the Lasso method, which simultaneously performs variables selection and regression coefficients estimation Yi corresponds to levels of 8-ohdg, augmentation index, and patella lead levels for each participant i and c is a non-negative tuning parameter. P1 and P2 represent the numbers of gene variants that could follow a dominant and recessive model, respectively. P3 corresponds to the number of gene variants that were deletions. The Lasso problem can be rewritten in the equivalent Lagrangian form in which the regression coefficients solved:λ is a non-negative parameter that controls the amount of shrinkage: the larger the value of λ, the greater the amount of shrinkage. There is a one-to-one correspondence between λ and c. The latter constraint  based on the lowest Akaike Information Criterion (AIC). This approach created a reduced set of candidate gene variants related to each pathway. We adjusted for age at this stage to explain some variability of the instrumental outcomes, and thus to help the selection process to be more accurate. We conducted the analysis using the Least Angle Regression (“lars”) R package i was:Using the signs of the non-zero coefficients of the Lasso penalization, we summed up gene variants related to each pathway to construct three score variables representing the allelic profile of each participant. The score for each individual We then dichotomized these score variables using the median of each score's distribution and used these resulted binary variables to investigate effect modification. A high score corresponds to a higher allelic risk profile. Another score approach has been used successfully before to examine gene-environment by pathway interactions Tests = 3 exposures *3 pathways), we conducted some multiple testing corrections using Bonferroni adjustment.Because our analysis involved 9 tests (n2.5 and black carbon were significantly correlated (correlation coefficient of 0.72).At baseline, the median age of the study participants was 72 years . About 1rs1001179 (+), rs480575 (−), rs2071746 (+), rs5995098 (+), rs1800566 (+), rs2282679 (−), and rs3170633 (−). (+) and (−) represent the sign we used when we added these seven variants up to create the genetic score. Among the nine gene variants candidates for the endothelial function pathway, the Lasso approach chose four of them: rs12944039 (+), rs2072324 (+), rs2255929 (+), and rs1137933 (−). Similarly, among the nine gene variants candidates for the metal processing pathway, five were chosen by the Lasso penalization rs224572 (−), rs422982 (+), rs12227734 (−), rs1005559 (−), and rs1799945 (−). Tables S1 to S3 in rs numbers and their corresponding genes.Among the 18 gene variants candidates for the oxidative stress pathway, seven were chosen by the Lasso procedure: interaction = 0.04)  (interaction = 0.12), C-reactive protein (pinteraction = 0.02), and ICAM-1 (pinteraction = 0.08)  . An inte = 0.06) . We also = 0.08) . For insIn addition, we observed significant associations between black carbon and fibrinogen only among those with high genetic score for oxidative stress, endothelial dysfunction, and impaired metal processing  to 3. NuAfter adjusting for multiple comparisons using the Bonferroni correction, none of the interactions remained statistically significant.We developed a new method to investigate the role of air pollution interacting with genetic variation within relevant pathways. Our genetic score approach suggests oxidative stress, endothelial function, and metal processing as biological pathways that play a role in enhancing air pollution effects on intermediary CVD-related blood markers. We observed significant greater associations between one-week exposure to particulate air pollution and fibrinogen, C-reactive protein, and ICAM-1 among elderly participants with higher allelic risk profiles, as compared to the same associations among individuals with lower allelic risk profiles.10 and fibrinogen was modified by single-nucleotide polymorphisms (SNPs) within the fibrinogen genes Our results support previous findings that showed similar intermediate-term associations between air pollutants (main effects and gene-environment interactions) and markers of coagulation and inflammation The greater increases in coagulation and inflammatory markers observed among subgroups with higher allelic risk profiles happened after one-week exposure to particle number and black carbon, which are traffic-related air pollutants. In Boston, black carbon originates mostly from local emissions during the morning and afternoon rush hours, as well as from transported traffic emissions 2.5 mass In addition, while particle number was not associated with C-reactive protein in the overall sample 2−, H2O2, and ONOO−) and a biological system's ability to promptly detoxify intermediary reactive products or simply repair the resulting damage Our results also support previous studies suggesting oxidative stress as an intermediary pathway between air pollution and cardiovascular related events In our study, we observed a positive association between black carbon and fibrinogen only among participants with higher genetic risk profile related to endothelial function. There is some evidence that oxidative stress and impaired endothelial function are interrelated. Many of the genes implicated in diseases associated with vascular dysfunction, such as atherosclerosis, are oxidative stress-sensitive, suggesting that an imbalance in cellular oxidative stress is a critical underlying factor This novel score approach we developed requires relevant instrumental outcomes to perform the first selection stage. The Lasso procedure returns regression coefficients that can be used to create the score. Since our outcome instruments were specific pathway-related measurements but did not represent the entire pathway, we chose to use weights equal to 1 to construct the score from the selected gene variants. Researchers can decide which weights they prefer to use based on their instrumental outcomes. We assumed equal weights for each of the gene variants chosen by the Lasso penalization. This assumption may also be a limitation, as some of the genes polymorphisms might have different functional effects and magnitude. Our defined biological pathways were not inclusive of all genes which regulate oxidative stress, endothelial dysfunction, or impaired metal processing. Therefore, we cannot definitely rule out false negative findings. We found some strong evidence of interactions between genetic-pathway and traffic-related air pollutants, which have been measured with ambient local monitors. Although this personal urban exposure approximation is likely to induce some Berkson measurement error This new statistical approach extends our knowledge on known and potential genetic pathways between air pollution and cardiovascular outcomes by prospectively investigating genetic pathway-environment interactions. Our score approach, selecting the gene variants by pathway and penalizing them by a Lasso procedure, enables us to reduce the set of candidate gene variants within pathway, to test for gene-air pollution interactions by pathway, and to reduce multiple comparisons since the significance examination of the interaction is based on a one degree of freedom test. Pathway analysis borrows information from correlated genes that belong to the same pathway and provides results with increased power. Our results demonstrated that the association between air pollutants and cardiovascular-related biomarkers are likely to differ according to the genetic background.File S1Combined file of supporting information.(DOC)Click here for additional data file.


TCF4 which confer susceptibility to FECD. Here, we reportthe findings of our independent validation study for TCF4 usingthe largest FECD dataset to date .Logistic regression with sex as a covariate was performed for three geneticmodels: dominant (DOM), additive (ADD), and recessive (REC). We foundsignificant association with rs613872, the target marker reported by Baratzet al.(2010), for all three genetic models .To strengthen the association study, we also conducted a genome-wide linkagescan on 64 multiplex families, composed primarily of affected sibling pairs(ASPs), using both parametric and non-parametric two-point and multipointanalyses. The most significant linkage region localizes to chromosome 18 from69.94cM to 85.29cM, with a peak multipointHLOD = 2.5 at rs1145315 (75.58cM) under the DOMmodel, mapping 1.5 Mb proximal to rs613872. In summary, our study presentsevidence to support the role of the intronic TCF4 singlenucleotide polymorphism rs613872 in late-onset FECD through both association andlinkage studies.Fuchs endothelial corneal dystrophy (FECD) is a common, late-onset disorder ofthe corneal endothelium. Although progress has been made in understanding thegenetic basis of FECD by studying large families in which the phenotype istransmitted in an autosomal dominant fashion, a recently reported genome-wideassociation study identified common alleles at a locus on chromosome 18 near Fuchs endothelial corneal dystrophy (FECD), first described by Ernst Fuchs COL8A2 (MIM: 120252) on chromosome 1,in which two missense mutations, p.L450W and p.Q455V/Q455K, have been replicated inrare early-onset (before 40 years of age) FECD multigenerational families COL8A2 missensemutations have been identified in late-onset FECD patients, although their roleremains unclear due to conflicting results SLC4A11 (MIM:610206), which causes autosomal recessive congenital hereditary endothelialdystrophy  TCF8 (MIM: 609141), whose loss offunction causes posterior polymorphous corneal dystrophy  FECD is often inherited as an autosomal dominant trait FCD1), 18(FCD2), 5 (FCD3), and 9(FCD4) COL8A2. Neither our late-onset FECD families nor our large,multigenerational family replicated the four previous FECD loci(FCD1 to FCD4), but the number of familiesused in the study may have been underpowered to detect significant linkage.Several genome-wide linkage scans using large multigenerational families havereported FECD loci on chromosomes 13  showed genome-wide significant associationwith FECD.The first genome-wide association study (GWAS) for FECD was reported recently, usingan initial dataset of 130 unrelated cases and 260 unaffected controls genotyped withthe Illumina 370K BeadChip panel Here we present additional evidence for the presence of a FECD locus on chromosome18. We report the results from an expansion of our previous linkage study, as wellas an association study analyzing the association of rs613872 with FECD in a datasetcontaining 450 unrelated FECD cases and 340 unaffected controls, the largest sampleof FECD patients interrogated to date.Our study was performed in accordance with the Declaration of Helsinki and theinstitutional review boards at Duke University Medical Center and Johns HopkinsUniversity (JHU) specifically approved this study. Both FECD study sites, theCornea Clinics at Duke University Eye Center (DUEC) and the Wilmer Eye Instituteat JHU, obtained the appropriate institutional review board approval forresearch on human subjects prior to initiating subject recruitment, and allindividuals gave written, informed consent. The control subjects from the Dukeglaucoma genetics study were also recruited under the approval of the DukeInstitutional Review Board, and consented to allow their biological samples tobe used by other research studies.All FECD subjects underwent detailed ophthalmic examination, including slit lampbiomicroscopy, to determine FECD severity. Grading of disease severity wasdetermined using a slightly modified version of the Krachmer scaleclassification system Two Caucasian datasets were used for association and linkage studies. Theassociation dataset consisted of 450 unrelated FECD cases, each with a Krachmergrade ≥2, and 340 unaffected controls with age at enrollment of≥45 years old. The 450 unrelated cases used here were either probandsfrom the family dataset (see below) or singleton cases, all of whom wereascertained through the cornea clinic at DUEC. The unrelated controls were from26 unaffected married-in spouses from the DUEC family dataset and 314 controlsubjects from the glaucoma genetic study at DUEC The linkage dataset consisted of 64 multiplex families  containing a total of 215 subjects . Families were ascertained independently from the Cornea Clinics atDUEC or at the Wilmer Eye Institute at JHU. Demographic data, including age atenrollment and gender, for the individuals analyzed in the association andlinkage datasets are summarized in TFC4, themost significantly associated SNP in the GWAS performed by Baratz and colleaguesPTPRG on chromosome 3, which was significant but not at agenome-wide level in the Baratz GWAS, to examine possible replication in ourdataset. The two SNPs were genotyped with pre-designed TaqMan® allelicdiscrimination assays , which use unlabeled polymerase chain reaction (PCR) primersand two allele-specific probes containing the TaqMan® minor groovebinding group (MGB) probe and either a FAM™ and VIC® dye labelin a 384-well plate format. PCR reactions were performed with Taqman®Universal PCR Master Mix on the GeneAmp® PCR System 9700 , and the ABI7900HT Fast PCR System was used for reading allelic discrimination calls. Quality control (QC) samples,including two CEPH (Centre d'Etude du Polymorphisme Humain) pedigreeindividuals, one no-template sample, and two duplicate samples , were contained within each quadrant of each 384-well plate. These QCsamples were used to provide duplicate samples within one quadrant, acrossquadrants within one plate, and across plates. Results of the CEPH and QC samplegenotyping were compared to identify possible sample plating errors andgenotype-calling inconsistencies, and none were observed. A threshold of95% genotyping efficiency was required for submission to the analysisdatabase.Association analysis focused on the SNP rs613872 in Two Illumina  SNP linkage panels were used for genome-wide linkagemarker genotyping. Seventy-two of the 215 linkage samples were included in ourprevious linkage study Reproducibility of genotypes was examined for the replicate QC samples located ineach 384-well Taqman plate to assess genotyping quality. Reproducibility ratesof 100% were observed. The two SNP markers, rs613872 and rs10490775,were examined for possible deviations from Hardy-Weinberg equilibrium (HWE).We instituted several QC measures to determine the final set of markers foranalysis. We first used the Illumina BeadStudio program to check genotypereproducibility rates using the replicated samples, family relationships, genderstatus of each DNA sample using Y chromosome markers, sample genotype call rates>99%, and the GenCall score which is a quality measure foreach genotype used in the Illumina genotyping system. In particular, the GenCallscore measures how close a genotype is to the center of the cluster of othersamples assigned the same genotype, as compared with the centers of the clustersof the other genotypes, and ranges from 0 to 1. The higher the GenCall score themore reliable the genotype. A set of clean markers was chosen based on GenCallscores ≥0.15, as well as meeting the other QC measures describedpreviously, for further data cleaning.For additional data quality assurance, 1000 markers with approximately equalinter-marker distances across the genome were selected to examine familyrelationships using the RELPAIR p-value for each marker All SNPs were tested for deviations from HWE. We randomly chose one affectedindividual per family to include in an unrelated affected dataset, and oneunaffected individual per family was selected to include in an unrelated,unaffected dataset. An exact test implemented in the Genetic Data Analysis (GDA)program was used to test HWE, by performing 3,200 permutations to estimate theempirical Genotypes of rs613872 (T and G nucleotide polymorphism) were tested using threegenetic models: dominant (DOM), additive (ADD), and recessive (REC). Thegenotypes of  were coded as  for DOM;  forADD; and  for REC where allele G is the rare allele. A logisticregression association analysis in PLINK http://linkage.rockefeller.edu/, provided in the public domainby Rockefeller University, New York, NY) were performed to generateheterogeneity logarithm of the odds (HLOD) scores. Since the mode of inheritanceis unknown for FECD, both DOM and REC genetic models were assumed in theparametric analysis with disease allele frequencies of 0.001 and 0.01respectively. The penetrance  was based on the assumed genetic models,and was consistent with the goal of conducting an affecteds-only parametriclinkage analysis.Two-point parametric linkage analyses using the FASTLINK 2)between markers at 0.16 in MERLIN to ensure independence between markers formultipoint linkage analysis.MERLIN P = 0.60), indicating highquality genotyping results. However, we found strong deviation from HWE for thismarker in the FECD case group (P<0.001). This may be thesource of the highly significant association signal we observed in our datasetfor all three genetic models .The effect size of the minor (risk) allele (G) in our dataset was strong: (1)DOM: odds ratio (OR) = 8.01, confidenceinterval (CI) of OR = ; (2) ADDmodel: OR = 5.35,CI =  ; (3) REC:OR = 4.06,CI =  . These results areconsistent with the findings of Baratz and colleagues PTPRG on chromosome 3, we did not detectsignificant association with FECD .We tested rs613872 in our case-control association dataset of 450 cases and 340controls; P = 3×10−26;femaleP = 1.2×10−16).However, this result may not reflect the true level of association due to thesmall sample size, particularly for males. Furthermore, the results betweengenders should not be compared due to the unbalanced sample sizes. Among our 450cases, we have 235 cases with grading information. We did not find thecorrelation between disease severity and genotypes of rs613872(P = 0.13). However, more samples may be neededto make a solid conclusion.When we examined gender-specific groups, we observed the same elevation of theheterozygous (GT) genotype in cases relative to controls in both genders . The SNPAfter implementing QC measures for the linkage dataset we removed fiveindividuals from three families due to relationship errors that could not beresolved and five individuals due to low sample call rates(<95%). In total, we analyzed 215 individuals in 64 families under the DOM model. The nonparametric analysis showedconsistent results for this region with a peak multipointHLOD = 1.48 at the same peak marker  thatappear to influence familial FECD TCF4 and FECD in the first genome-wide associationstudy carried out for FECD TCF4, rs613872, with highly significant allelic and genotypicp-values of 2.34×10−26 and1.29×10−18, respectively, in their combinedanalysis of their small discovery and replication datasets.To date, progress towards identifying the genetic underpinnings of FECD has beenlimited to a handful of genes, including p-value of9.33×10−35 (DOM). Additionally, we show that thebest multipoint linkage region from our family dataset is also located on chromosome18 (68.94 cM to 85.29 cM), with a peak multipointHLOD = 2.5 at rs1145315, 1.5 Mb away from rs613872in TCF4. The consistent findings in both our linkage andassociation studies for an association of rs613872 with FECD together with thefindings of Baratz et al. suggest that the association is probablynot spurious but, rather, is due to certain recombination events in this region thatmay increase susceptibility to FECD. Additionally, the excess of heterozygousgenotypes at rs613872 in both our cases and those of Baratz et al.warrants further investigation. Although we replicated the association of rs613872with FECD in our dataset, we failed to replicate the association with rs10490775 inPTPRG. Since we also did not detect evidence of linkage tochromosome 3, we hypothesize that either this locus does not influence FECD risk orits effect on FECD risk is small.Here, we present a large case-control association dataset that replicates theserecent genome-wide association data It is of great interest to know if rs613872 genotypes can predict disease severity.Our 235 cases with grading information did not show significant correlation to thegenotypes of rs613872, which is consistent to the finding from Riazuddin et al.et al. are of Europeandescent, it will be important to examine whether the association between rs613872and FECD risk replicates in other ethnic and racial populations. It is possible thatalternative TCF4 risk alleles may be associated in other ethnic andracial populations, which would argue for this locus being functionally important inFECD pathogenesis. Alternatively, if the true FECD disease variant is in LD withrs613872, a lack of association in other ethnic and racial populations may helpexplain why the disorder has a lower prevalence in other populations like African,East Asian, and South American populations.According to data from the Human Genome Diversity Project, the minor (risk) allele,G, of rs613872 is rare to nonexistent in populations from Africa, Eastern Asia, andCentral and South America, yet is more frequent in European, Middle Eastern, andSouthern Asian populations . Given tTCF4 genethat might help explain the functional mechanism behind the association of rs613872with FECD, so clearly further studies are needed to detect such variations if theyexist. Although TCF4 is an attractive candidate gene within theFCD2 locus, much remains unknown. If TCF4plays a role in familial FECD, further research is needed todiscover the mutation(s)within TCF4 that segregate with disease in families that showevidence of linkage to FCD2. l. Until such a causative mutation(s)is identified, restraint needs to be exercised in not drawing premature conclusionsthat a causal link between the TCF4 protein and FECD has been identified.GWAS studies commonly raise the question of how to interpret the biologicalsignificance of statistically associated SNPs, which may be located in intronic orintergenic regions with no obvious connection to the disease phenotype. Baratz andcolleagues did not find any coding variations within the TCF4 is expressed in eyetissues, particularly in the corneal endothelium Tcf4 mutant mice do not appear to contain vision/eyeabnormalities as a phenotypic feature Ini1(SMARCB1) and Brg1 (SMARCA4).These are both components of the SWI/SNF chromatin remodeling complex that isrequired for transcriptional regulation of genes normally repressed by chromatinTCF4 is a transcription factor, it is possible thatvariation at this site might have an effect on the spatiotemporal expression ofTCF4 and, in turn, affect the expression of some of itstargets. Further studies are required to investigate the veracity thishypothesis.Expression data have indicated that Our study provides supporting evidence of a linkage signal to the strong associationresults of rs613872. This not only further confirms the importance of a locus onchromosome 18 in influencing FECD risk, but also indicates that a causal variant forFECD may be identified within this locus. It is highly possible that rs613872 istagging a rare variant within this locus that plays a role in the etiology of FECD.Additional sequencing studies using family datasets that are highly linked to thislocus is a potential strategy to identify the biological mechanisms underlying howthis locus influences FECD pathogenesis.


Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (<30 ng/mL) determination of antigen-specific antibodies in ten minutes of total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy with an area under curve (AUC of 98% was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies. We propose that the presented framework could find use in convenient and cost-efficient quality control of antibody production, as well as in providing a platform for multiplexed affinity-based assays in low-resource or mobile settings. Protein microarrays are widely used tools for diagnostic biomarker analysis, with proven utility in such varied fields of human medicine as for instance autoimmunity, allergy, infection and cancer . In partThe assay procedures for protein microarrays are low- to moderately labor-intensive, and may due to the ambient-analyte conditions governing the microspot assay dynamics offer excellent sensitivity on par with ELISAs . HoweverManual handling and advanced detection strategies result in assay times normally spanning several hours and consequently current microarray technologies are not well suited for mobile, on-site or point of care applications. While it is likely that a large proportion of microarray analyses could preferentially continue to be performed within advanced health care sites such as hospitals or centralized laboratories, portable microarray solutions may support more rapid clinical decision making in emergency situations in the field or in such areas where little healthcare infrastructure exists. Further, point of care microarray tests might serve to relieve health care resources when patients or care-givers are able to conduct regular testing themselves. Finally, such portable tests could facilitate those unable to go to a hospital or health care point for testing .While there is a wide variety of simple portable tests commercially available, including for instance rapid tests for sexually transmitted diseases, cardiovascular disease and allergies, such tests are typically simplex or low-plex, with limited quantitative ability and sensitivity, and may further suffer in diagnostic accuracy . Thus, cA considerable hurdle for creating portable, easy to use planar microarray solutions appears to be the accommodation of many sequential assay steps on-chip, as well as the need for comprehensive sample mixing in order to avoid long assay times due to large diffusion coefficients of protein biomarkers. These challenges are largely avoided in immunochromatographic/lateral flow-based tests, and to this end we previously demonstrated the feasibility of combining a lateral flow/dipstick assay framework commonly used in simplex/low-plex rapid tests with protein microarray patterning for rapid and accurate determination of levels of bovine IgG raised towards mycoplasma infection  using a In this paper, we expand the concept into a general high-plexed lateral flow protein microarray (LFM) strategy, with 384 protein fragments (approximately 80–100 aa)  where thIn this work, by employing a prototype lateral flow protein microarray device, we were able to accurately determine levels of 26 individual HPA antibodies, as well as up to 8 concurrent specific IgG analytes in approximately 10 min total assay time. We argue that: (a) the demonstrated concordance with an established glass/fluorescence microarray assay; (b) the simplicity of the assay procedure drawing from the benefits of common lateral flow/dipstick tests; (c) the highly multiplexed capacity of the assay; (d) the short assay time; and (e) the convenient read-out options, make the presented assay framework an interesting tool for rapid quality control of raised antibody reagents, and may further find use in translating the capabilities of protein microarrays to point of care/low resource settings.In this study, a novel protein antigen lateral flow microarray containing 384 different capture microspots patterned onto a nitrocellulose substrate and with a gold nanobead detection strategy  was evalA selection of 26 purified rabbit IgG isolates, diluted to 0.17–6.91 μg/mL was analyzed in triplicates on the lateral flow microarray as well as on the reference glass slide microarray . It was A good agreement between the lateral flow array and the glass array results was found  and the A moderate correlation coefficient is to be expected due to differences in substrate coupling to the protein antigens, as well as substantial differences in assay dynamics. However, the sensitive and specific binding of the sample antibodies on the corresponding antigen microspots should be retained, and this is indeed what the ROC analysis demonstrates.Two antibodies  were selected for a sensitivity analysis. Each antibody was diluted 1:100, 1:1000, 1:10.000, 1:100.000 in assay buffer, and three replicates per antibody were run for each dilution. As compared with the negative control, it was found that the sensitivity could be estimated to around 30 ng/mL or better and that the response tended to be log-linear .From the triplicate runs of the total cohort of 26 specific IgG species included in this study (and under the assumption that the uncontrolled variation was Gaussian distributed), we calculated the standard deviation of each analyzed antibody. We found that the average coefficient of variation (%CV) was 13% [5%–21%]. The level of variability may impede highly resolved quantitative analysis, but could still be acceptable for semi-quantitative approaches.In order to demonstrate the array’s capability of detecting an increasing number of antibody analytes, we created mixtures of 2, 2, 4 and 8 antibodies to be analyzed on the lateral flow microarray. It was found that the array could accurately identify all the constituents of the mixes, although the positive signal to noise ratios gradually decreased  due to i6-albumin binding protein domain [E. coli and the protein product subsequently affinity purified under denaturing conditions before being immunized into rabbits to stimulate antibody production. The antibodies are harvested from the rabbit after 4 months and purified by means of affinity chromatography using the immunogens/antigens as affinity ligands [All protein antigens were selected from the antibody quality control division of the HPA project . The antn domain . Antigen ligands .Protein antigens were diluted in printing buffer  before patterning on substrates. The assay buffer used for most dilutions and washes contained phosphate buffered saline (PBS) + 0.5% Tween20, together with 3% bovine serum albumin (Sigma) and 1% sucrose (Sigma) at pH 7.4.Cardboard-backed nitrocellulose membranes  were cut into 12 by 25 mm strips and glued to 0.8 mm thick arraying slides (Arrayit) with off-the-shelf super glue . 384 individual protein antigen capture probes were then spotted onto the membranes using an Arrayjet Marathon (Arrayjet Ltd.) at 80 μg/mL in printing buffer. The array blocks were printed in a 16 by 24 spot layout with 280 μm distance between spot centers . ApproxiThe patterning of glass microarrays was carried out using the same printing protocol as for the lateral flow microarray, but using epoxy-derivatized glass slides  as substrates. After printing, slides were allowed to rest at 37 °C for 24 h, after which slides were blocked in PBS + 0.1% Tween20 + 3% BSA for 1 h on a shaker at 160 rpm. Slides were then washed three times with PBS for 5 min each, followed by brief rinsing in deionized water and finally drying by spinning 2 × 3 min at 700 rpm. The assay procedure for the use of glass slide antigen arrays in the analysis and quality control of rabbit sera has been described elsewhere . Briefly6ABP-tag present on all antigens  was applied 2 mm from the top end of each strip across the width of the membrane, forming the lower boundary of a sample drop-in area. The resulting hydrophobic barrier forced the sample to travel only through and not on top of the nitrocellulose membrane. A cotton sheet (Whatman) of around 1 × 2 cm was placed at the end of the membrane to serve as a fluid sink. Initially, the membrane was presoaked with 30 μL assay buffer in order to avoid non-specific binding and provide a homogeneous flow profile. Subsequently, 30 μL antibody sample was applied, followed by a 15 μL wash with assay buffer. Next, 30 μL of biotin-conjugated goat anti-rabbit F(ab′)2 (Jackson Immunoresearch) was applied, again followed by a 15 μL wash step. Finally, 30 μL of OD10 40 nm monoclonal goat anti-biotin coated gold particles  diluted 1:3 in assay buffer was applied, followed by a 30 μL wash step. Each applied liquid step needed around 90 seconds to complete, giving a total assay time of around 10 minutes. As a quality control step to ensure all antigen spots had been printed satisfactorily, a 100 μg/mL rabbit anti-HisABP antibody (generated in house from HPA project) was applied to one strip instead of rabbit IgG solution, which resulted in binding to the Hisantigens . In all After drying (1–5 min depending on the surrounding temperature and humidity), the arrays were scanned using a standard tabletop HP Scanjet 8270 (Hewlett Packard) at 1200 dpi. 16 bit gray scale tiff images were acquired by VueScan 8.6.34 (Hamrick Software) and subsequently inverted and analyzed using GenePix Pro 5.1 (Axon Instruments). Data analysis was performed with the statistical language R version 2.12 . The medEfforts to discover novel clinically useful biomarkers is continuously generating better molecular tools that through earlier and more accurate diagnosis and better disease monitoring may eventually greatly reduce the global burden of disease . A key c


The inhibitory output from the internal pallidum and substantia nigra to the thalamus forms an important link in the transmission of basal ganglia processing to cortex. Two hypotheses consider either inhibition of thalamic activity or thalamic excitation via post-inhibitory rebound burst firing as the functional mode of this link. We used optogenetics to characterize the synaptic properties of nigral input to motor thalamus in adult mouse brain slices, and to determine in what conditions the nigral inhibition of motor thalamus is transmitted via inhibition or rebound firing. Our results are more consistent with graded inhibition of spiking for conditions expected in normal awake animals, because inhibitory potentials from nigral input were generally not sufficient to elicit rebound spikes when the thalamic neurons were actively firing. However, with bursty or fast trains of nigral input low-threshold rebound spike bursts could be triggered for low levels of excitation. This may form the basis of pathological burst generation and transmission in parkinsonian conditions. The brain was removed quickly and slices cut in ice-cold solution at 250 μm with a vibratome (Microm HM 650). Slices were incubated in extracellular medium (choline or sucrose replaced with NaCl 124) for 60–90 min at 34°C and afterward gradually returned to room temperature. Despite these precautions viable slices were only obtained from about 50% of perfused mice, as adult mouse thalamic tissue poses a great challenge due to extensive myelination.All animal procedures followed approved Emory University IACUC protocols. Adult male C57BL/6 mice (Jackson Labs) of 3–7 months of age were stereotactically injected with AAV5-Syn-ChR2(H134R)-EYFP or AAV1-Syn-ChR2(H134R)-EYFP  into the SNr pars reticulata under isoflurane anesthesia. At 3–5 weeks following injection mice were sedated in isoflurane and anesthetized with ketamine/xylazine (200 mg/kg ketamine and 40 mg/kg xylazine i.p.). After complete cessation of reflexes mice were perfused transcardially with cold extracellular saline modified to protect from excitotoxic processes following hypoxia. The media contained (in mM): (Sucrose 235.4 (or Choline Chloride 117.7); KCl 2.5; NaHin vivo, due to desensitization and the relatively slow deactivation time constant of ChR2  . Pipettes were filled with solution containing (in mM): 140 K-gluconate, 6 NaCl, 2 MgCl2, 4 Na2ATP, 0.4 Na3GTP, 0.2 EGTA, 5 glutathione, 0.5 spermine, 0.02 Alexa-568, and 10 HEPES, pH 7.3 with KOH. The temperature of the slice chamber was maintained at 32°C with a feedback controller and a Peltier element . Optical stimulation of ChR2 was performed either with a blue laser light source  through a 200 μm glass fiber pointed at the motor thalamus at a distance of 1–3 mm or with whole field light flashes at 60× magnification with the fluorescent imaging light source of our microscope  filtered to pass blue light. In the second case a Uniblitz shutter was used to control exposure times. In some cases both light sources were used on the same recorded neurons and the elicited IPSPs were found to be equivalent. However, sub-millisecond exposure times were only possible using the laser light source. We used single light pulses as well as paired pulses and light pulse trains of up to 20 Hz frequency for stimulation in order to explore post-inhibitory effects after periods of inhibition of varying duration. While faster than 20 Hz stimulation might more naturally mimic SNr firing frequencies In order to selectively stimulate nigral inputs to motor thalamus during whole cell recordings of VM neurons we injected an AAV vector carrying YFP-tagged channelrhodopsin-2 (ChR2) into the SNr of adult mice 3–5 weeks prior to preparing brain slices . In horizontal brain slices YFP was clearly visible in the SNr on the side of the injection, in connecting fiber bundles, and in motor thalamus Figure . Whole cStimulation of ChR2 with blue light  at the location of whole cell recordings in thalamus reliably triggered an inhibitory post-synaptic potential (IPSP) in current clamp Figure . This IPN = 13) were confirmed by eliciting IPSPs with optical nigral terminal stimulation (see Methods). IZ neurons all showed a hyperpolarized non-spiking baseline, and typically required a current injection of 100–300 pA to elicit a tonic single-spike mode . Repetitive shorter negative current injection pulses of 50 ms resulted in a train of LTS bursts . At hyperpolarized membrane potentials the driving force of inhibition was too small to allow substantial further hyperpolarization , IPSPs, and dendritic T-type channels in the control of rebound LTS activity. To directly test for such interactions, we applied low-pressure, sustained glutamate puffs  to the dendritic area of IZ neurons during whole cell recording to trigger receptor-evoked spiking. This method very reliably evoked spike trains of several seconds duration Figure . The spiN = 5 of 5 neurons tested). As also observed in whole cell recordings, in a subpopulation of neurons (2 of 5 neurons tested) trains of optogenetic light stimulation could evoke an LTS burst from rest .A recent report in songbird motor thalamus indicates that basal ganglia input in vitro study need to be considered when interpreting the data with respect to in vivo function of thalamic coding of basal ganglia input. First, due to slicing procedures, slice recovery, lack of neuromodulation in vitro, partial deafferentation, altered chemical milieu, etc, synaptic properties may be different from normal thalamic processing in the awake mouse. These are limitations inherent in all slice work, and extrapolation to the awake condition should be best understood as a hypothesis to be examined in the awake condition when possible. Second, the recordings obtained in our study were not of sufficient numbers, and the anatomical specification of recording sites was not accurate enough, to assess the potential for heterogeneity between different sub-areas of the IZ thalamic zone that can be expected on anatomical grounds  of thalamus in rat  in order to avoid describing a transient developmental state of this pathway, as GABA receptor subtypes undergo developmental regulation (Fritschy et al., A IPSC decay kinetics based on a different subunit composition (Huntsman and Huguenard, The average decay time of the IPSCs observed in IZ neurons in the present study was 14 ms, compared to 9 ms in the VB thalamus of mouse pups and 3.4 ms in the VB thalamus of adult mice (Peden et al., A key question our study addressed was whether the transmission of nigral output to cerebral cortex via IZ thalamus involves post-inhibitory rebound LTS bursts as has been observed with basal ganglia inhibition of songbird motor thalamus (Person and Perkel, In our preparation it was also unusual to elicit a post-inhibitory rebound from the hyperpolarized resting potential Figure , becausein vivo could shut down glutamate elicited spiking completely (Figures We did find that optical nigral stimulation reliably induced a short pause in IZ thalamic firing, and that light pulse trains simulating a period of increased nigral activity  Figures , 7. In c Figures , however Figures  and 66 H Figures . On this Figures . Similar Figures . Motor t Figures . Based o Figures , 2009.Thalamic activity in normal animals is not entirely devoid of LTS bursts Sherman, , howeverJeremy R. Edgerton obtained slice recordings in cell attached and whole cell mode, analyzed data, and critically edited the manuscript. Dieter Jaeger obtained slice recordings in whole cell mode, analyzed data, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Crebbp+/− FL HSCs had less unprocessed pre-mRNA without a corresponding reduction in total mRNA levels. Among the genes thus identified were the key regulators of HSC function Itga4, Msi2 and Tcf4. A similar but much weaker effect was apparent in Ep300+/− FL HSCs, indicating that, in this context as in others, the two paralogs are not interchangeable. As a group, the down-regulated intronic probe sets could discriminate adult HSCs from more mature cell types, suggesting that the underlying mechanism is regulated with differentiation stage and is active in both fetal and adult hematopoiesis. Consistent with increased myelopoiesis in Crebbp hemizygous mice, targeted reduction of CREBBP abundance by shRNA in the multipotent EML cell line triggered spontaneous myeloid differentiation in the absence of the normally required inductive signals. In addition, differences in protein levels between phenotypically distinct EML subpopulations were better predicted by taking into account not only the total mRNA signal but also the amount of unspliced message present. CREBBP thus appears to selectively influence the timing and degree of pre-mRNA processing of genes essential for HSC regulation and thereby has the potential to alter subsequent cell fate decisions in HSCs.Global expression analysis of fetal liver hematopoietic stem cells (FL HSCs) revealed the presence of unspliced pre-mRNA for a number of genes in normal FL HSCs. In a subset of these genes,  Crebbp are essential for HSC self-renewal while Ep300+/− HSCs are not compromised in this respectCrebbp gene results in multi-lineage defects in differentiation with a clear excess in myeloid cell production and an age-dependent increase in the incidence of hematologic malignanciesCyclic-AMP-responsive element binding protein (CREB) binding protein (CREBBP) – more commonly referred to as CBP – is a multifunctional protein which facilitates gene expression through several mechanisms, including chromatin remodeling, acetylation of associated proteins, and recruitment of the basal transcription machinery to promoters CREBBP also acts as a scaffold in numerous protein-protein interactions In addition to its activities as coactivator and integrator, confocal microscopy studies have localized CREBBP to nuclear speckles containing splicing proteinsCrebbp+/− FL HSCs relative to wild-type (WT) mapped entirely within introns, rather than detecting exonic or spliced sequences. We therefore set out to test whether this might be evidence that reduced CREBBP levels selectively alter the generation of full-length, unspliced pre-mRNA. We also asked whether this process might be associated with differentiation since self-renewal and lineage commitment are both responses for which HSCs are primed.The presence of unspliced transcripts in HSCs and the links between CREBBP and EP300 with the constitutive production of unspliced RNA and with pre-mRNA processing prompted us to examine more closely an anomaly we had noted in microarray-based gene expression studies but had previously attributed to experimental “noise”. We had noticed that more than half of the probe sets down-regulated in Crebbp+/− FL HSCs relative to WT by quantitative RT-PCR (qRT). In addition to primary FL HSCs, we looked for CREBBP-associated changes in mRNA, intronic message and protein levels in multipotent EML cells which retain lymphoid, myeloid and erythroid potential in the presence of Stem Cell Factor (SCF) but can be induced to undergo differentiation by treatment with appropriate stimuliCrebbp by shRNA in EML cells was sufficient to trigger widespread myeloid differentiation of EML cells, bypassing their usual requirement for withdrawal of SCF and treatment with retinoic acid, interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF). A subset of genes tested also showed altered levels of intronic message in subpopulations of EML cells at different phenotypically-defined stages of development which corresponded to changes in protein abundance not predicted by full-length mRNA levels. Furthermore, the differences in intronic levels correlated with differentiation stage-dependent changes in CREBBP levels. Taken together, our data suggest a novel, cell type-specific function for CREBBP in regulating the timing and extent of pre-mRNA splicing of key regulators of HSC maintenance and function.As predicted by our microarray studies, we found that several genes associated with HSC function showed variable ratios of intronic to total RNA signal in Crebbp+/− FL HSCs. Our in-house annotation of the Affymetrix Mouse 430 2.0 microarrays (see 2 test p <1.1×10−15). Of the 83 intronic probe sets representing 77 distinct transcripts (both protein-coding and non-coding genes), 13 were designed to detect expressed sequence tags (ESTs) in libraries constructed from adult HSCsMbnl1 and Meis1. In both cases, the mRNA probe set could detect either a putative (non-RefSeq) alternative 3′ exon or an intron in a longer isoform.We have carried out a global analysis of expression profiles of WT and rays see . This isEp300+/− FL HSC expression data set we produced concurrently in which 15/35 differentially expressed probe set were intronic (χ2 test p = 1.3×10−5), again all of them down-regulated. In contrast, probe sets altered in Crebbp+/− mouse embryonic fibroblasts (MEFs) relative to WT MEFs showed no enrichment for intronic probe sets (Cip1/Waf1) is required for HSC self-renewal and its loss leads to exhaustion of the HSC pool upon serial transplantationCrebbp+/− FL HSCs, however, FL HSCs null for Cdkn1a showed both up- and down-regulated intronic probe sets at a frequency close to predicted relative to their WT controls . In the absence of appropriate data sets from fetal liver, we selected expression series comparing adult bone marrow (BM) HSCs and various populations of early progenitors and more mature hematopoietic cellsTo determine whether these differentially expressed intronic probe sets were functionally relevant, we first asked whether the intronic probe sets down-regulated in Crebbp+/− FL HSCs. Only 0.52% of the random samples yielded such a clean distinction of HSCs from mature cell types, arguing against the separation occurring purely by chance and suggesting that the underlying process may be common to both adult and fetal HSCs.We used either all 83 probe sets for clustering or the single most highly expressed probe set per transcript  to avoid overweighting genes detected by multiple intronic probe sets. The cell populations clustered the same way with both approaches. Crebbp+/− vs WT FL HSC data set was retained. Mds1, marked by a red asterisk in both Are the intronic probe sets simply surrogate measures of the total mRNA level? For each gene with at least one mRNA probe set (74/77 genes), the one with the largest difference in the Hierarchical clustering of 2 other published data sets were carried out in the same manner . In eachRbpms, for its implication in developmental processesCrebbp+/− samples. For intronic amplicons, the reproducibility of the results depended on the proximity of the amplicon to poly(A) tracts of sufficient length to potentially prime the oligo(dT) reverse transcriptionTcf4/E2-2 and Msi2 intronic amplicons both showed reduced levels in Crebbp+/− HSC relative to WT. Two other genes, Itga4 and Rbpms, which had down-regulated intronic probe sets  also exhibited greater reduction in intronic than total signal. Crebbp+/− (A) or Ep300+/− (B). Genes for which the ratio is similar for total mRNA and unspliced transcripts fall between the vertical and horizontal grey lines marking 1.5-fold differences. Tcf4, Msi2 and Rbpms, on the other hand, fall within the vertical lines, indicating similar mRNA levels in WT and Crebbp+/− HSCs, but lie well above the horizontal line marking a greater than 1.5-fold excess intronic signal in WT cells. Itga4 mRNA levels decreased roughly 2-fold in Crebbp+/− HSCs relative to WT but unspliced transcript levels are reduced even more substantially (∼10-fold). Consistent with the array data, Ep300+/− samples showed attenuated, if any, difference in intronic and total mRNA ratios . In some cases, both intronic and total signal changed proportionately  while in others the variation was greater in the intronic signal than in total mRNA level . The change in intronic signal relative to total message thus seems gene-specific rather than a function of length or mRNA abundance . Overall, the clustering and qRT results indicate that the mechanism underlying the alterations in intronic signal is both cell-type specific and selective in its targets.Many of our target genes were >100 kilobases long and their mRNA was detected at modest levels on the microarrays. Consequently, we wondered whether changes in intronic sequence detection was a widespread phenomenon and whether it correlated with transcript length or expression level. We therefore carried out a further set of qRT reactions using linked primer pairs for which either the forward or the reverse primer was common to both the exonic and intronic amplicons for a given gene  proximal to their transcription start sites  relative to the exonic target genes  clones were isolated in SCF-containing methylcellulose and then passaged in liquid culture in the presence of SCF (i.e. under non-differentiating conditions). Within 3–4 weeks, 39% of clones were dead or dying, 39% had differentiated and the remainder underwent extensive cell death before subclones emerged, either looking and growing like the parental EML cells or else differentiating. Crebbp message (as measured by qRT at week 5) were more likely to differentiate and eventually die. Clones that survived for the full length of the experiment (120 days) generally grew in suspension like parental EML cells and seemed to arise as subclones in cultures that had undergone substantial cell death. As controls, we also followed 20 clones receiving control shRNA targeting GFP and 20 that were cloned but not exposed to virus. All grew like parental EML cells for more than a month, indicating that differentiation and death were not predominantly side-effects of lentiviral treatment or cloning.Forty Crebbp knock-down, reminiscent of the increased myelopoiesis seen in Crebbp+/− animalsDown-regulation of CREBBP in EML cells thus mimics the mix of cell death and myeloid differentiation seen in these cells when they are driven to differentiate by ATRA, IL-3 and GM-CSF stimulationItga4, Msi2 and Tcf4, each of which has been implicated in hematopoietic developmentCrebbp+/− FL HSCs relative to WT  we are detecting varies by target gene, cell type and differentiation state.Crebbp expression develop myelodysplastic disease within their first year of life (manuscript submitted) and IRF1 has also been implicated in the pathophysiology of immune-mediated marrow failure in myelodysplastic syndromeA further indication that the changes in intron levels we found in FL HSCs are functionally relevant is the fact that the transcription factor binding sites enriched in the proximal promoter regions of intronic genes were in part different from those for genes in which total mRNA abundance changes. Binding sites for SOX17, a factor essential for fetal but not adult hematopoiesisInterestingly, binding sites for the Ets family factors SFPI1/PU.1, ETS1, GABPA and ELK1 were enriched in down-regulated intronic targets and in the up-regulated mRNA set but not in down-regulated mRNA targets. This shared regulation by up-regulated mRNAs and transcripts with reduced intronic levels would make sense if, as our data suggests, a reduction in intronic signal results in greater mRNA splicing leading to increased protein production. Shared promoter elements would thus allow coordinated expression by directly up-regulating transcription for some targets while increasing the processing of unspliced transcripts for others. This partitioning of transcription factor binding site clusters between intronic and mRNA targets is worthy of further study as it points to specific regulatory modules participating in CREBBP-mediated control of mRNA processing.Crebbp expression pushes multipotent EML cells to commit to myelopoiesis. The HSC-like EML cell line was established by introducing a dominant-negative retinoic acid receptor (RARα403) into mouse BM. Due to an RARα403 block, EML cells require treatment with ATRA to restore their ability to produce granulocyte-macrophage progenitors (CFU-GM) in response to IL-3 and GM-CSFCrebbp expression levels, however, allowed EML cells to bypass this block and differentiate into macrophages even in the presence of SCF doses normally able to maintain the cells in an undifferentiated state. This spontaneous myeloid differentiation of EML cells after Crebbp knock-down is reminiscent of the increased myelopoiesis seen in Crebbp+/− animalsIt is still unclear whether the link between altered intronic signal and differentiation is causative but what is clear from our data is that knocking down We have shown here that both splicing changes and spontaneous myeloid differentiation are sensitive to CREBBP dosage and selective alterations in abundance of intronic sequences correlate with differentiation in EML cells and changes in protein levels. Splicing changes varied by cell type and gene but nevertheless appear regulated as opposed to being a surrogate measure of total mRNA. Our data are consistent with a bookmarking model of HSC lineage priming with CREBBP involved in multiple steps of the lineage decision-making process as coactivator of transcription and coregulator of pre-mRNA processing. They also fit well with the concept that key regulators such as CREBBP can alter the landscape of HSC fate decisionsCrebbp+/− and Ep300+/− mice  are fully back-crossed on a C57BL/6 background. WT littermates served as controls. B6;129S2-tm1TyjCdkn1a/J mice and B6129SF2 WT controls were purchased from Jackson Laboratories .Mice were bred and maintained under pathogen-free conditions at the animal facility of the GCCRI. All animal procedures were approved by the University Health Science Center Institutional Animal Care and Use Committee (protocol numbers 06030 and 06059). 6 cells, indicating that the embryos were at a similar stage of development when sacrificedMated females were checked daily for the presence of vaginal copulation plugs, which defined day 0.5 of gestation. Plugged females were sacrificed on day 14.5 of gestation and their embryos harvested. The average cell count of 21 WT fetal livers (14 litters) was 21.3±4.1×10Single cell suspensions were prepared from each FL and viably frozen. Once enough embryos of each genotype were collected, cells were thawed and prepared for HSC isolation. Lineage negative  SCA1+ AA4.1+ KIT++−5M β-mercaptoethanol, penicillin/streptomycin; Invitrogen, Carlsbad, CA).Day 14.5 post-coitus embryo bodies were disaggregated in 0.25% trypsin and plated in MEF medium  or for which the interquartile range was <0.5. In the case of Crebbp+/−, Ep300+/− and their WT control FL HSCs, the values of the technical replicates were averaged prior to filtering based on minimum intensity levels and variation. Paired Student T-tests with p-value <0.05 and a fold-change >1.5 were used as the cut-off for calling significant change.All data is MIAME-compliant and the arrays are available for download from the Gene Expression Omnibus (GSE27987). For each HSC sample, 12 ng of RNA were amplified using the Ovation RNA amplification kit . MEF RNA was isolated using TRIzol (Invitrogen) and used without amplification. After biotin labeling, samples were hybridized to Affymetrix Gene Chip Mouse Genome 430 2.0 microarrays. Each microarray was checked for large-scale defects, RNA degradation and the proportion of probe sets called “Present” or “Absent” using MAS5 . All arrays were comparable and satisfactory by these metrics. Arrays for each group were normalized and corrected for background using the BioconductorProbe sets on the arrays used in these studies typically comprise 11 25-mers designed to interrogate GenBank mRNA and EST sequences. We used Blasthttp://www.ncbi.nlm.nih.gov/geo/) and either reprocessed from image files (GSE17765Crebbp+/− vs WT HSCs and (b) the most variable mRNA probe set for each gene corresponding to an intronic probe set in (a). GenePatternhclust and cutree functions from the R stats packageData sets of hematopoietic populations using the same microarray platform as our studies were downloaded from the Gene Expression Omnibus  were obtained from ATCC . Cells were cultured in IMDM medium supplemented with 20% FBS  and rmSCF  to maintain them in an undifferentiated state . Myeloid differentiation was induced by a 3-step protocol adapted from a previous study5′-CAAGCACTGGGAATTCTCT-3′ in mouse Crebbp (shCrebbp) was created by cloning oligonucleotides into the FSIPPW vector as previously described5′-AAGAACGGCATCAAGGTGAACTT-3′) was generated similarly. Both were packaged as previously describedA lentivirus-encoded shRNA targeting the sequence Phenotypically primitive EML subpopulations−ΔΔCt relative quantification methodGapdh. Primers were designed using the Primer3 web interfacehttp://frodo.wi.mit.edu/primer3/). See Total RNA from cloned EML cells was isolated by TRIzol extraction and purified with the Qiagen RNeasy kit . Total RNA from all other samples was extracted using the Qiagen RNeasy (Plus) Micro Kit as directed by the manufacturer. RNA was then reverse transcribed using the High Capacity cDNA Reverse Transcription Kit . FL HSC was amplified using the Ovation RNA amplification kit. PCR was performed using either the SYBR Green PCR kit (Qiagen) or the GoTaq qPCR Master Mix  on a 7500 Real-Time PCR System (Applied Biosciences). Data were analyzed using the 2Anti-CREBBP antibody AC26Whole cell lysates from 15,000 cells of each phenotype were suspended in RIPA buffer containing protease inhibitors  and PMSF  then loaded in each well after heat denaturation (10 min @ 70°C).5 cells suspended in RIPA buffer with protease inhibitors and PMSF or 12.5 µg of nuclear extracts were loaded per well, as indicated.Whole cell lysates from 4–5×10http://rsb.info.nih.gov/ij) was used to quantify band intensities. Significance levels for the differences between cell types were determined by correlated-samples one-way ANOVA and Tukey HSD test using the web-based calculator at VassarStats: Website for Statistical Computation (http://faculty.vassar.edu/lowry/anova1u.html).In all cases, ImageJ software Click here for additional data file.Table S2Quantitative RT-PCR Validation of Microarray Results. Detailed Excel table of locations of primer sequences relative to microarray probe sets and qRT validation results for exonic and intronic targets. For intronic probe sets, the table also includes the length of potential poly(A) RT priming sites and predicted presence of intronic poly(A) signals.(XLS)Click here for additional data file.


DUSP1 gene encodes a member of the dual-specificity phosphatase family previously identified as being differentially expressed in visceral adipose tissue (VAT) of severely obese men with versus without the metabolic syndrome. Objective. To test the association between DUSP1 polymorphisms, obesity-related metabolic complications, gene methylation, and expression levels in VAT. Methods. The DUSP1 locus and promoter region were sequenced in 25 individuals. SNPs were tested for association with obesity-related complications in a cohort of more than 1900 severely obese individuals. The impact of SNPs on methylation levels of 36 CpG sites and correlations between DNA methylation and gene expression levels in VAT were computed in a subset of 14 samples. Results. Heterozygotes for rs881150 had lower HDL-cholesterol levels , and homozygotes for the minor allele of rs13184134 and rs7702178 had increased fasting glucose levels . rs881150 was associated with methylation levels of CpG sites located ~1250 bp upstream the transcription start site. Methylation levels of 4 CpG sites were inversely correlated with DUSP1 gene expression. Conclusion. These results suggest that DUSP1 polymorphisms modulate plasma glucose and HDL-C levels in obese patients possibly through alterations of DNA methylation and gene expression levels.The  DUSP1 gene.Obesity in adults and in adolescents significantly increases the risk for several chronic diseases including cardiovascular diseases (CVD) and type 2 diabetes (T2D) , 2. ExceDUSP1 gene expression have been reported in various disorders. DUSP1 overexpression has been reported in breast, gastric, and lung cancers [DUSP1 with concomitant promoter hypermethylation and loss of heterozygosity at the DUSP1 locus was reported in HCC [DUSP1 expression was suggested in prostate cancer [DUSP1, also known as MKP1, is a member of the dual-specificity phosphatase family involved in the regulation of the innate immune function . Followi cancers –17. In c cancers –21. Downd in HCC , and epie cancer .DUSP1 gene variations may contribute to partially explain interindividual variability observed in obesity-related metabolic complication among obese individuals through the regulation of gene methylation and expression. An extensive molecular analysis of the human DUSP1 gene was undertaken to test for associations between DUSP1 polymorphisms and obesity-related metabolic complications in a cohort of severely obese patients undergoing a bariatric surgery. The impact of the SNPs associated with metabolic complications on gene methylation and expression in VAT was thereafter analyzed.Based on the functions of DUSP1 in innate immune function, inflammation, and apoptosis and its implication in various disorders and differential expression we have shown in VAT of MetS+ versus MetS− obese individuals , we postN = 597) and women (N = 1309) have been recruited. These Caucasian patients from the eastern part of the Quebec province, Canada, underwent biliopancreatic diversion with duodenal switch at the Quebec Cardiology and Pulmonology Institute . The study was approved by the Université Laval ethics committee, and all individuals provided written informed consent before their inclusion. The surgical protocol is presented elsewhere [Between June 2000 and July 2012, 1906 severely obese men (lsewhere . Body welsewhere . Body ma2) not taking any medication to treat MetS features were selected from our cohort for gene expression profiling using oligonucleotide microarrays. Seven of them were affected by the MetS (MetS+ group) and were matched for age, BMI, and smoking status to obese individuals without MetS . Characteristics of these individuals stratified by MetS group as well as procedures for expression profiling are available elsewhere [DUSP1 gene was identified as part of a list of 489 genes differentially expressed in VAT between the two groups.In a previous study , a subselsewhere . FollowiDUSP1 gene expression was measured by real-time PCR (RT-PCR)  in the same 14 adipose tissue samples selected for the original microarray experiment [GAPDH housekeeping gene . Relative quantification estimations were performed on an Applied Biosystems 7500 Fast Real-Time PCR System following the manufacturer's recommendations, and the ΔΔCT calculation method was used to evaluate the mean fold expression difference (MFED) between MetS− and MetS+ groups.To validate the microarray results, periment  using cuDUSP1 gene were first amplified and sequenced with primers designed using human DUSP1 public sequence (accession number: NC_000005) in a subset of 25 severely obese men  including the 14 individuals selected for gene expression profiling. Additional 11 individuals were randomly selected. MetS status was taken into account to include similar proportions of MetS+ and MetS− individuals for DUSP1 sequencing. The BigDye Terminator 3.1 kit was used for sequencing, and samples were run on an ABI 3730/XL DNA Analyzer automated sequencer (Applied Biosystems). From the SNPs identified by direct sequencing, five tagging single nucleotide polymorphisms (tSNP)  were selected using the tagger selection algorithm of the Haploview software [Genomic DNA was extracted from the blood buffy coat using the GenElute Blood Genomic DNA kit . The promoter region (~1500 bp), exons, and intronic flanking regions of the software , 25. SelDUSP1 promoter region were performed on a subset of 91 consecutively recruited individuals from our cohort (4.8% of the whole cohort). Degree of concordance between TaqMan genotyping and sequencing results was calculated.Validation of genotyping data was conducted for the SNP rs88150 demonstrating overrepresentation of rare homozygote individuals. PCR amplification and direct sequencing of μg of DNA, and quantitative DNA methylation analysis was carried out at the McGill University and Génome Québec Innovation Centre . Infinium HumanMethylation450 BeadChip  was processed according to the manufacturer's instructions. The BeadChip interrogates more than 485000 methylation sites at single-nucleotide resolution. Methylation data was visualized and analyzed using the GenomeStudio software version 2011.1 (Illumina Inc.) and the methylation module. Methylation levels  were estimated as the ratio of signal intensity of the methylated alleles to the sum of methylated and unmethylated intensity signals of the alleles ). Data correction  was applied using internal control probe pairs. CpG sites with a detection P value > 0.05 were removed from analysis. Methylation ratios  of CpG sites located within the DUSP1 locus and promoter region (~2 kb upstream transcription start site (TSS)) were extracted using the GenomeStudio Methylation Module, thus leading to a total of 36 CpG sites analyzed in this study.DNA methylation analysis was conducted on the 14 obese men initially selected from our cohort for gene expression profiling. Genomic DNA was extracted from 200 mg of VAT using the DNeasy Blood & Tissue kit , as recommended by the manufacturer. Bisulfite conversion was conducted on 1 t-test to identify differentially expressed genes between MetS+ and MetS− groups. Hardy-Weinberg equilibrium (HWE), linkage disequilibrium (r2), and selection of tSNPs  were conducted using the Haploview software [t-tests. Pairwise Pearson correlations between methylation and expression levels and between microarray and RT-PCR data were computed. Statistical significance was defined as P ≤ 0.05. P values presented are for transformed and adjusted values when appropriate. Statistical analyses were performed using SAS software version 9.2 .Phenotypic data  were reported as mean ± SD. Gene expression microarray analysis was conducted using unpaired Student's software  and the software . After gThe potential functional impact of CVD risk factor-associated SNPs was assessed using two different programs. The MatInspector program was used to analyze the impact of promoter SNPs on transcription factors binding sites , 27 whiln = 1549) or MetS− (n = 350 or 18.4%). As shown in The present study included 1906 severely obese individuals (597 men and 1309 women) undergoing bariatric surgery who were consecutively recruited. From these individuals, 1899 were successfully classified either as MetS+  revealed 489 differentially expressed genes between MetS+ and MetS− groups [ DUSP1 gene was significantly underexpressed  in VAT of MetS+ versus MetS− obese men. Validation of microarray data for the DUSP1 gene was conducted in the same subset of individuals using RT-PCR. According to RT-PCR results, the DUSP1 gene was found to be underexpressed in VAT of MetS+ obese men . DUSP1 gene expression levels obtained by RT-PCR were highly correlated to those obtained by microarrays . More details on RT-PCR and microarray results are available in Supplementary Table 1 available online at http://dx.doi.org/10.1155/2013/609748.As previously reported, microarray analysis of 14 obese men  and the rs201026723 (p.Met60Ile) were further genotyped in the whole cohort of 1906 severely obese individuals. The number of genotypes and HWE P values obtained are shown in Promoter region, exons, and intronic flanking sequences of the P = 0.01), with the heterozygotes exhibiting lower levels , homozygotes for the minor allele of rs13184134 or rs7702178 having increased fasting glucose levels , and genotype groups were tested. Following adjustment for the effects of age, sex, and BMI, rs881150 was associated with plasma HDL-C levels . Prediction models have been used to assess the potential impact of CVD risk factor-associated SNPs. Analysis of the potential effect of the rs7702178 intronic SNP on splicing revealed that it slightly reduces the strength of an acceptor site located on the reverse strand (0.53 to 0.47), thus unlikely impacting on mRNA splicing for which junction between intron 2 and exon 3 scored 0.88. The MatInspector program was used to analyze the impact of the two CVD risk factor-associated promoter SNPs  on binding of transcription factors. Both SNPs demonstrated potential impact (disruption or creation) on transcription factor binding sites based on matrix similarities (Supplementary Table 2). These two promoter variants could thus result in differences in gene expression and contribute to the difference of DUSP1 SNPs and cardiometabolic risk factors, associations between CVD risk factor-associated SNPs  and gene methylation levels were tested. Of the 36 CpG sites analyzed, 25 were located within the promoter region, 8 in exons, and 3 in introns. The list of CpG sites analyzed with corresponding localization is provided in Supplementary Table 3. Significant associations between CpG site methylation levels and genotype groups were identified for 10 CpG sites. A trend towards an association (P < 0.10) was also observed for 5 CpG sites. As shown in r = 0.642) was found between these 7 CpG sites. The link between gene methylation and gene expression was then assessed for SNP-associated CpG sites. From the 10 SNP-associated CpG sites analyzed, 4 were inversely correlated with gene expression levels while a trend toward a significant association was identified for 3 other sites .To explore potential mechanisms underlying the relation between er sites . FurtherDUSP1 gene expression have been reported in various types of cancers [DUSP1 in VAT of severely obese individuals with the MetS, thus extending the potential pathophysiological importance of this gene to obesity-related metabolic disease. Following DUSP1 sequencing and identification of tSNPs, associations of DUSP1 SNPs with CVD risk factors were identified, namely, HDL-C and fasting glucose levels. To provide potential explanation for these associations, further analyses were conducted on the three CVD risk factor-associated SNPs . Results presented here suggest that DUSP1 SNPs modulate plasma glucose and HDL-C levels in severely obese patients and that this effect is potentially mediated through alterations of gene methylation and expression.Over the past years, differences in  cancers –21. The DUSP1 gene in the interindividual variability observed in obesity-related metabolic complications. A gene dosage effect of DUSP1 on IL-10 levels has been reported in response to lipopolysaccharide at the systemic level in mice and on macrophage stimulation [DUSP1 in VAT may have a physiological impact.Our analysis of differentially expressed genes in VAT of severely obese individuals with and without MetS revealed a potential implication of the mulation . This suWith genetic variations potentially having an impact on gene expression, assessment of SNPs was conducted and associations between selected SNPs and CVD risk factors  were tested. Two SNPs (rs13184134 and rs7702178) were found to be associated with fasting glucose levels with a third one associated with HDL-C levels. DUSP1 is known to be involved in the regulation of innate immune function , inflammDUSP1 gene expression levels and the associations identified for rs881150, rs13184134, and rs7702178. With SNPs having an impact on the binding of transcription factors with consequent impact on transcriptional activity [DUSP1 promoter SNPs may potentially affect the binding of transcription factors and promoter transcriptional activity. With SNPs potentially having an impact on gene methylation leading to differences in gene expression [DUSP1 gene methylation and expression levels, it is tempting to speculate that associations identified here between DUSP1 SNPs and cardiometabolic risk factors may be mediated through differences in CpG sites methylation and expression.Further analyses were performed to better understand the potential underlying mechanism for the difference in activity  and geneactivity , the firpression , 39, thepression , 41. In N = 7; MetS− group, N = 7) [DUSP1 resequencing and tSNPs selection were conducted in 25 individuals including the highly selected subset. Associations were thereafter tested in the whole cohort of 1906 individuals to identify SNPs explaining interindividual variability in obesity-related metabolic complications among severely obese patients. Looking at SNPs having a small impact on the phenotypes of interest, we deliberately chose to exclude testing the association of rare SNPs with obesity-related metabolic complications as our study had a limited power. Results presented here demonstrate an impact of DUSP1 gene variations on obesity-related complications. Although the impact is of small effect size, these results might partially explain interindividual variability observed in metabolic complications among obese individuals and thus highlight a potential new MetS candidate gene. The present findings were obtained in a cohort of severely obese individuals. Because inflammation is found in severely obese individuals, the impact of DUSP1 SNPs through altered regulation of the innate immune function may be subjected to different modulations. Thus, results from the present study cannot be extrapolated to individuals with less pronounced obesity levels or to the general population. It would be relevant to conduct similar studies in the general population or in a cohort of normal weight individuals showing low or moderate levels of inflammation.The current study has some limitations. Gene expression and DNA methylation analyses were conducted in a relatively small number of selected individuals from our larger cohort. However, individuals from this subset were free of metabolic diseases such as type 2 diabetes, cardiomyopathy, or endocrine disorders and were matched for age, BMI, and smoking status to patients with, on average, none of the recognized features of the MetS except high waist girth  . Gene exDUSP1 promoter methylation and gene expression levels argue for an impact of DUSP1 promoter SNPs on gene expression possibly mediated through alterations in specific CpG sites methylation levels. Combined with the associations identified here between SNPs and cardiometabolic risk factors, these results provide a potential novel contributor to the modulation of plasma glucose and HDL-C levels in obese patients. It would be interesting to identify pathways mediating the impact of DUSP1 SNPs and to investigate how epigenetic differences at the human DUSP1 locus relate to the large-interindividual variability observed in metabolic complications among obese individuals.In summary, the results obtained from the analysis of Supplementary Table 1. Validation of microarray results by real-time PCR for the DUSP1 gene.Supplementary Table 2. Potential impact of CVD-associated promoter SNPs on transcription factor binding sites.Supplementary Table 3. List of CpG sites analyzed with corresponding localization.Supplementary Figure 1. Schematic representation of sequence-derived SNPs at the DUSP1 locus (chromosome 5q34).Click here for additional data file.