Supercentenarians (110 years or older) are the world’s oldest people. Seventy four are alive worldwide, with twenty two in the United States. We performed whole-genome sequencing on 17 supercentenarians to explore the genetic basis underlying extreme human longevity. We found no significant evidence of enrichment for a single rare protein-altering variant or for a gene harboring different rare protein altering variants in supercentenarian compared to control genomes. We followed up on the gene most enriched for rare protein-altering variants in our cohort of supercentenarians, TSHZ3, by sequencing it in a second cohort of 99 long-lived individuals but did not find a significant enrichment. The genome of one supercentenarian had a pathogenic mutation in DSC2, known to predispose to arrhythmogenic right ventricular cardiomyopathy, which is recommended to be reported to this individual as an incidental finding according to a recent position statement by the American College of Medical Genetics and Genomics. Even with this pathogenic mutation, the proband lived to over 110 years. The entire list of rare protein-altering variants and DNA sequence of all 17 supercentenarian genomes is available as a resource to assist the discovery of the genetic basis of extreme longevity in future studies. Supercentenarians are the world’s oldest people, living beyond 110 years of age The genetic component of human lifespan based on twin studies has been estimated to be around 20–30 percent in the normal population Several gene association studies have compared cohorts of long-lived subjects to controls. Analysis of candidate genes has shown that polymorphisms in the Insulin-like Growth Factor 1 Receptor gene (IGF1R) and the FOXO3 transcription factor gene are associated with extreme longevity More recently, studies have begun to use whole-exome sequencing and whole-genome sequencing (WGS) of centenarians to find variants associated with extreme longevity Here, we have sequenced the genomes of 17 supercentenarians. We limited the majority of our analyses to the thirteen genomes from Caucasian females. From this small sample size, we were unable to find rare protein-altering variants significantly associated with extreme longevity. However, we did find that one supercentenarian carries a pathogenic variant associated with arrhythmogenic right ventricular cardiomyopathy (ARVC), which had little or no effect on his/her health as this person lived over 110 years.Supercentenarian subjects, their family members, or their caretakers provided written informed consent. The study was approved by the Stanford University Institutional Review Board (IRB-19119) and by the Western Institutional Review Board (WIRB protocol #20101350). Supercentenarians were considered validated  if they possessed each of the following documents: (1) A birth certificate, a baptismal certificate, or Census Record dating back to the original time of birth; (2) A marriage certificate in the case of married women not using their maiden names; (3) a current government-issued photo ID, such as a driver’s license or passport. Supercentenarian health status and medical history for major age-related diseases were based on interviews conducted with subjects and/or their caretakers.Whole-blood samples were drawn into PAXgene (Qiagen) blood tubes from which high molecular weight DNA was isolated. DNA samples were quantified using a dsDNA Broad-Range Assay on a Qubit Fluorometer (Life Technologies) and checked for size and degradation on an agarose gel. For Sanger sequencing, samples were amplified by nested PCR and variants were validated by forward and reverse reads. Primers were designed with Primer3 Principal component analysis (PCA) of ancestry was done by analyzing the intersection of all genotyped SNPs from 1184 individuals from 12 different populations from HapMap Phase 3 All DNA samples were submitted for WGS to 40x coverage by Complete Genomics, Inc. (CGI). Standard protocols were used to map reads and call variants using CGI pipeline 2.0.2 To filter out common variants, we used dbSNP version 131 Next, we applied a collapsing test to determine if any gene showed an enrichment of rare protein-altering variants in supercentenarian vs. control genomes. We started with the set of protein-altering variants in autosomal RefSeq genes observed in supercentenarians and controls (34 Caucasians from the PGP), and filtered to retain only rare variants with a minor allele frequency (MAF) <1.5% in 1000G EUR, and with an empirical MAF<10% in our samples. For each gene, we computed the RVT1 statistic Samples from the Georgia Centenarian Study We used the recently published list from the American College of Medical Genetics and Genomics (ACMG) of potentially lethal pathogenic variants in 56 genes recommended for reporting to subjects Upon acceptance for publication, the complete genome sequence for the 17 supercentenarians will be deposited in dbGAP and Google Genomics.We recruited 17 supercentenarians and validated their age of 110 years or greater see . Their mAmong the 17 supercentenarians, at least one subject had a previous case of cancer and one was diagnosed with Alzheimer’s disease. To the best of our knowledge, none of the supercentenarians were known to have cardiovascular disease, stroke or diabetes at the time of enrollment. In contrast, people in the US at age 85 often have had at least one major age-related disease. For example, 45 percent of 85-year olds have been diagnosed with cancer and 35 percent have had an incidence of cardiovascular disease We isolated DNA from whole blood and sent the samples to Complete Genomics for WGS. Samples were sequenced to a read depth of 40x, and 94.1% of the genomes and 94.8% of the exomes had a read depth of at least 20x . To confNext, we checked the genomes of our supercentenarians for unknown relatedness to each other, as any close relationship would confound analyses for enrichment of shared rare variants. We checked for shared regions of identity-by-descent using Maximum-likelihood Estimation of Recent Shared Ancestry −5 per birth −6. A variant that was present in four supercentenarian genomes but absent in all genomes in 1000G EUR would have a P-value of 7.4×10−07 and would have been detected by our method. Using high quality sequence calls, preliminary analysis suggested that one novel variant was shared by three supercentenarian subjects but not by the control genomes; however, Sanger sequencing subsequently showed that this was a sequencing error in the supercentenarian data. To increase our sensitivity for finding a longevity variant, we repeated the analysis including low quality calls. This yielded three additional novel variants in the supercentenarian genomes. However, Sanger sequencing showed that each of the three variants was a sequencing error. Even though the overall error rates for SNPs in WGS data (>40x coverage) are under 1% For people born around 1900, the odds of living to 110 are estimated to be less than 10Another possibility is that there may be a gene that confers extreme longevity when it is altered by any one of a number of protein alterations. Many of the supercentenarians may carry variants in the same gene, but the variant in each supercentenarian may be different. The variants could act in a dominant fashion and affect only one of the two alleles. Or else they could act in a recessive fashion such that both alleles would be affected, either with the same variant (homozygous) or with different mutations in each allele (compound heterozygous). Therefore, we asked whether any of the genes in the female Caucasian supercentenarian genomes was enriched for harboring rare protein-altering variants (either one or two copies) when compared to control genomes. Although the 1000G are a large group of controls, they cannot be used for a gene-based test as only the frequency of each variant is known, and not the individual genotypes. Therefore, as controls we used WGS of 34 Caucasian individuals (ages 21–79) from the Personal Genome Project (PGP) that were sequenced on the same platform as the supercentenarians We created a pipeline that used the annotated supercentenarian and PGP genomes from the previous analyses as input see also . Next, w−06 to correct for multiple hypothesis testing. We were thus powered to detect genes altered in seven supercentenarians, if the gene harbored no alleles in any of the 34 controls. None of the genes showed a genome-wide significant enrichment using the Bonferroni threshold , but controls would only carry zero or one mutation, but not two. The RVT1 test performs a logistic regression on the proportion of rare variants and hence might detect a bias in supercentenarians  vs. the controls . But the RVT1 test was not specifically designed to compare the number of compound heterozygous cases and controls. We performed a gene-based test to compare the number of cases and controls carrying at least two variants in the same gene applying Fisher’s Exact Test to compute P values. We found that no gene was significantly enriched for two or more mutations after multiple testing correction .−4). For SCN11A, three control genomes contained rare protein-altering variants with minor allele frequencies below 0.05 (but above 0.01). This result weakens the distinction between the supercentenarian genomes and the control genomes, and thus this gene was discarded from further analysis. NAB2 was discarded when Sanger sequencing showed that two out of four variants were sequencing errors. For TSHZ3, Sanger sequencing validated all four protein-altering variants, and this gene was chosen as a candidate for follow-up experiments.Although none of the genes showed a significant enrichment in the female Caucasian supercentenarian genomes, we nevertheless decided to follow up on the top three genes from the RVT1 burden test: TSHZ3, NAB2, and SCN11A  or ClinVar. The first supercentenarian carried a missense SNP (L1564P) in the Breast Cancer Associated 1 (BRCA1) gene. Although null mutations in BRCA1 are pathogenic, the pathogenicity of L1564P is unclear. The L1564P variant appeared in the breast cancer of a 33-year old female along with another missense SNP (Q1785H) The second supercentenarian possessed a known pathogenic SNP (rs397514042) that disrupts a splice-site in Desmocollin-2 (DSC2). Desmocollin-2 is part of the myocardial desmosome structure in the heart. Loss-of-function mutations in DSC2 and other genes of the desmosome are associated with Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) The rs397514042 SNP has been observed in two patients with ARVC We have sequenced the genomes of 17 supercentenarians (over 110 years of age) to see if we could uncover the genetic basis for their extreme longevity. We analyzed rare protein-altering variants, but found no strong evidence for enrichment of either a single variant or a single gene harboring different variants in female Caucasian supercentenarians compared to controls. From our gene-based analysis, the gene showing the most enrichment for protein-altering variants in supercentenarians compared to controls was the TSHZ3 transcription-factor gene. Because it was the top hit, we pursued this gene further in a study consisting of 99 genomes from subjects aged 98–105 years old. We found that TSHZ3 carried protein-altering variants in more of the long-lived subjects than the controls, although this difference was not statistically significant (P = 0.17).A larger sample size would be required to establish whether the difference in frequency of protein-altering variants in TSHZ3 between subjects with extreme longevity compared to controls is statistically significant. We did not analyze single nucleotide variants in non-coding DNA in the supercentenarians because of the large number of non-coding variants compared to coding variants. Our analysis of putative rare protein-altering variants in the whole genome sequencing data led us to test a number of candidates, of which 30% were subsequently determined to be false positive variant calls in WGS data. This high false discovery rate is consistent with previous reports Our analyses show that it is extremely unlikely that there is a single gene harboring rare protein-altering variants shared by all supercentenarians but no controls. It is not surprising that a highly complex trait such as longevity is not explained by a single Mendelian gene.To our surprise, we discovered that one of our supercentenarians carried a known pathogenic allele in the DSC2 gene associated with arrhythmogenic right ventricular cardiomyopathy (ARVC). This is a potentially fatal condition, causing affected individuals to die of sudden cardiac death. This example points out an important aspect about policy regarding the reporting of pathogenic mutations found in genomic sequences. The American College of Medical Genetics and Genomics identified a set of genes that can cause pathology when disrupted. But what is often not known is how frequently people with the variant have pathology . Our example shows that the DSC2 pathogenic mutation rs397514042 did not cause a fatal cardiomyopathy during the proband’s over 110 years of life. Thus, the presence of this mutation in the DNA sequence of a young person today should be reported to him/her and their families with caution, as it may or may not result in arrhythmogenic right ventricular cardiomyopathy. Generally, variants that are annotated as pathogenic are of unknown penetrance The full set of protein-coding variants are given in Figure S1Genome coverage for supercentenarians. Average genome coverage is shown for the whole genome (dark grey) and exome (light grey) of all 17 supercentenarians. Coverage is shown for ≥1x and ≥20x coverage.(PDF)Click here for additional data file.Figure S2Principal Component Analysis of supercentenarian ancestry. PCA was performed on all 17 supercentenarians (black dots) and HapMap genotypes. All Caucasian supercentenarians (CAU) clustered with Caucasian HapMap individuals, while the two supercentenarians of Hispanic ethnicity clustered with Mexican HapMap individuals and the African-American supercentenarian (AA) clustered with African HapMap individuals. HapMap populations are ASW (African ancestry in Southwest USA), CEU (Utah residents with Northern and Western European ancestry from the CEPH collection), CHB , CHD , GIH , JPT , LWK , MXL , MKK , TSI  and YRI . See insert for color codes.(PDF)Click here for additional data file.Table S1Baseline statistics for 34 Caucasian PGP genomes.(XLSX)Click here for additional data file.Table S2All variants in protein coding regions with genotypes for all 17 supercentenarian and 34 PGP control genomes.(XLSX)Click here for additional data file.Table S3Burden of rare protein-altering variants per gene in supercentenarians and controls.(XLSX)Click here for additional data file.Table S4Gene-based Fisher’s Exact test for recessive model of rare protein-altering variants in supercentenarians and controls.(XLSX)Click here for additional data file.


Early surgical intervention in children with drug resistant epilepsy has benefits but requires using tolerable and minimally invasive tests. EEG-fMRI studies have demonstrated good sensitivity for the localization of epileptic focus but a poor yield although the reasons for this have not been systematically addressed. While adults EEG-fMRI studies are performed in the “resting state”; children are commonly sedated however, this has associated risks and potential confounds. In this study, we assessed the impact of the following factors on the tolerability and results of EEG-fMRI in children: viewing a movie inside the scanner; movement; occurrence of interictal epileptiform discharges (IED); scan duration and design efficiency. This work’s motivation is to optimize EEG-fMRI parameters to make this test widely available to paediatric populationForty-six children with focal epilepsy and 20 controls (6–18) underwent EEG-fMRI. For two 10 minutes sessions subjects were told to lie still with eyes closed, as it is classically performed in adult studies (“rest sessions”), for another two sessions, subjects watched a child friendly stimulation i.e. movie (“movie sessions”). IED were mapped with EEG-fMRI for each session and across sessions. The resulting maps were classified as concordant/discordant with the presumed epileptogenic focus for each subject.Movement increased with scan duration, but the movie reduced movement by ~40% when played within the first 20 minutes. There was no effect of movie on the occurrence of IED, nor in the concordance of the test. Ability of EEG-fMRI to map the epileptogenic region was similar for the 20 and 40 minute scan durations. Design efficiency was predictive of concordance.A child friendly natural stimulus improves the tolerability of EEG-fMRI and reduces in-scanner movement without having an effect on IED occurrence and quality of EEG-fMRI maps. This allowed us to scan children as young as 6 and obtain localising information without sedation. Our data suggest that ~20 minutes is the optimal length of scanning for EEG-fMRI studies in children with frequent IED. The efficiency of the fMRI design derived from spontaneous IED generation is an important factor for producing concordant results. EEG-fMRI has shown over the last decade to be a useful technique to map epileptic networks across a range of epilepsy syndromes –3. It prHowever EEG-fMRI studies have a number of potential limitations that may play a role in the sensitivity of this test to map the epileptogenic region. These include:Low yield: Due to the unpredictable nature of IED Long duration: In order to maximize the chances of capturing IED EEG-fMRI studies tend to use long scanning times. The optimal length for EEG-fMRI studies has not been formally assessed.Lack of motion control: associated with long scanning timesVariability and poor definition of baseline. Differently from cognitive fMRI studies, EEG-fMRI baseline is unconstrained and this may affect the power of the test.There are no studies systematically addressing these interacting factors that affect EEG-fMRI sensitivity and tolerabilityA large proportion of the epileptic syndromes develop during infancy and childhood . From thOne of the main potential limitations of MRI in a paediatric population in general is the effects of movement on data quality and the tolerance of children to the test. Adult’s long resting state EEG-fMRI protocols are unlikely to be widely tolerable in a paediatric population. Most previous EEG-fMRI studies in children have used sedation to control movement inside the scanner , 22. HowNatural stimulation has been proposed as a way to improve compliance and reduce movement as well as to create a more comfortable experience in the scanner for paediatric populations . NaturalThe effects of low level sensory stimulation such as a movies on epileptic activity are unknown as is the impact on the functional maps derived from EEG-fMRI studies. To our knowledge, no previous EEG-fMRI study has used nor investigated this approach.In this study, we aim to test the impact of: a) a child friendly natural stimulus (i.e. movie); b) in-scanner head movement; c)the occurrence of interictal epileptiform discharges (IED); d) scan duration; e) design efficiency; on the results of EEG-fMRI for IED localisation in children with drug-resistant focal epilepsy.The study was approved by NRES Committee London—Surrey Borders Research Ethics Committee (REC) London Centre. REC reference: 11/LO/1421 Written informed consent was obtained from all the participants’ guardians in the participant’s behalf.46 children  with drug resistant epilepsy underwent simultaneous EEG-fMRI.Recruited patients were undergoing evaluation for resective epilepsy surgery at Great Ormond Street Hospital . Inclusion criteria for the study were: the presence of frequent interictal epileptiform discharges on EEG and participant ages between 6 and 18. Additionally, a group of 20 healthy volunteers  with an age between 6.6–16.7 years and a mean age of 11.5 years were scanned following the same protocol.Controls were significantly younger than patients in our population. We controlled for this effect statistically when investigating the effect of natural stimulation on in-scanner movement by using age as a confound.All patients underwent detailed clinical evaluation, prolonged video telemetry-EEG to document seizures and structural MRI. Additional tests including FDG-PET, ictal SPECT, MEG and intracranial EEG were performed in those patients in whom additional information for the localisation of the epileptogenic region was required. The localization of the epileptogenic region was determined by consensus between a group of expert epileptologists based on the review of the results of the panel of diagnostic tests. Clinical discussion to determine the epileptogenic region takes place at a multidisciplinary meeting that included neurology, neurosurgery, neurophysiology, neuroradiology and neuropsychology specialists. See Participants were fitted with a 64-electrode MRI-compatible EEG cap . Preparation commenced 1 hour before the scanning time. Prior to placing the subject in the MRI scanner, scalp EEG was recorded for 10 minutes to obtain a baseline EEG.Subjects were then transferred to the MRI scanner table. The head was immobilized using a vacuum and foam cushions. Subjects watched a TV screen via a mirror mounted on the head coil and sound was delivered through a set of MRI compatible headphones which supressed scanner acquisition noise.The subjects were videoed inside the scanner with an MRI compatible camera  interfaced with Brain Products recorder software.The MRI protocol consisted of a T1-weighted volume scan followed by 4 10-minute fMRI sessions. For two of the fMRI sessions, the subjects were presented with a movie (a ‘Tom and Jerry’ cartoon clip) chosen to attract and maintain their attention (‘movie session’) and for the other two sessions, the subjects were instructed to rest with their eyes closed (‘rest session’). The movie presentation was configured as a paradigm with two blocks of cartoon lasting for 4 minutes each interleaved with 1.2 minute blocks where the screen displayed the words “please wait”. Rest and movie sessions were alternated and the participants were randomly allocated to start with the cartoon or resting session.To investigate the tolerance of children to EEG-fMRI, subjects were asked after each session whether they were comfortable and if they would like to proceed with another session until they completed a maximum of 4 sessions.Tolerance of our study population to EEG-fMRI is expressed as the fraction of patients that completed a certain proportion of the full protocol (measured as the number of completed sessions). Correlation between the age of the subject and the number of sessions tolerated was investigated by comparing the mean ages of subjects completing or failing to complete the full protocol using a T-test. Statistical tests were calculated using SPSS software version 21.Subjects were scanned in a 1.5T Siemens Avanto scanner at Great Ormond Street Hospital  with a 12 channel receive head coil. Echo planar imaging (EPI) parameters are as follows: 3.3x3.3x4mm resolution with a field of view (FOV) = 210mm, TR = 2160ms, TE = 30ms, flip angle = 75 degrees, number of slices = 30, slice thickness = 3mm, slice gap = 1mm, and matrix 64x64, with 300 volumes and a voxel size of 3.3 x 3.3x 4.0 mm. Image acquisition was continuous.T1-weighted whole-brain structural images were also obtained in all subjects using a Fast Low Angle Single Shot (FLASH) gradient echo sequence.EEG data was acquired with a 64 electrode MRI-compatible equipment  simultaneously with MRI. Data was band-pass filtered at 0.016Hz-1 kHz with 16-bit digitalization (0.5μV resolution) and sampling rate was set to 5 kHz.MR gradient and pulse-related artefacts were removed from the EEG using template artefact subtraction , 26 implEach of the EEGs was reviewed in both bipolar and referential montages for the accurate identification of epileptiform abnormalities.EEG-fMRI analysis to determine the presence of regional IED-related BOLD changes was performed only for the patients and not for the controls.http://www.fil.ion.ucl.ac.uk/spm/spm8). For this purpose, IEDs onsets and durations were converted into a temporal regressor, and convolved with the canonical hemodynamic response function (HRF) and its temporal and dispersion derivatives, resulting in 3 regressors for each event type [The fMRI time series were analysed using a general linear model in SPM8 . Changes in BOLD signal were considered significant above a threshold with p<0.001 (uncorrected) and a cluster size voxel with a minimum of 5 contiguous voxels [EEG-fMRI maps were classified as concordant if they contained a cluster of significant BOLD signal change within the presumed epileptogenic region and discordant otherwise , 5, 28. We analysed the effect of movie on the following variables:The effect of the movie on subject movement was estimated using a regression model. The dependant variable was movement inside the scanner which was quantified by mean frame-wise displacement (MFD) which is the difference in the position between each volume measured by spatial realignment in spm8 (see above). The MFD was transformed using the natural logarithm to normalize the distribution of the residuals. The independent variables were the session type , time (session order 1 to 4) and age. The interaction between session type and time was also explored.The model parameters were estimated using a mixed effects model in R . This moWe compared the rate of IEDs during the movie and the rest sessions across all the patients. We further divided movie sessions into the different blocks of the paradigm (cartoon and “please wait” blocks) to account for potential differences in IED rate during the stimulus period. An ANOVA was used to assess the impact of condition on the occurrence of IEDsEEG-fMRI maps were created for each session and the concordance of maps was assessed as detailed above. The effect of movie on the concordance of EEG-fMRI maps was tested using a two tailed chi square test of session type (movie/rest)*concordance of EEG-fMRI maps (SPSS version 21).To explore the effect of EEG-fMRI scan duration in our population, we calculated the proportion of maps with concordant/discordant results obtained when the first session was considered alone and compared this proportion to the concordance rate obtained when 2 sessions (20 minutes), three sessions (30 minutes) and four sessions (40 minutes) were considered. For this purpose, individual general lineal models were generated with either one; two; three or four sessions for each subject using spm8. Models were otherwise specified as described in the EEG-fMRI analysis section above.IED frequency and distribution should affect the sensitivity of EEG-fMRI to detect IED-related fMRI responses, and therefore explain the results in terms of concordance (here discordance includes a null result). It is necessary to quantify this effect in terms of the efficiency of the model to detect fMRI changes, that can be estimated as for cognitive fMRI paradigms using the design efficiency  which es68 children were recruited, of which 66 (46 patients and 20 controls) underwent EEG-fMRI. 83.3% (55/66) of them completed the whole protocol (4 sessions), while 13.7% (9/66) and 3% (2/66) completed 3 and 2 sessions respectively. One of the participants only tolerated the scan when the movie was being displayed.There were 2 failed scans; one due to intolerance of the EEG-cap application and the second case due to fear of lying down in the scanner table and entering the scanner bore.Using a two sample t-test it was found there was a statistically significant difference between the age of the patients and the number of sessions that they tolerated. Patients who completed the 4 sessions had a mean age of 13.7 years compared to 9.6 years for those not completing the protocol (t(44) = 3.457, p< 0.05).The amount of movement inside the scanner was greater for sessions 2-3-4 relative to session 1, t(176) = 2.54, 2.72, 4.34 respectively  . Older sEighty percent of the patients (38/46) had spikes during the EEG-fMRI scan. For those patients that did not have detectable epileptic activity during the scan, it was absent for all sessions and independent of session type (movie or rest).The mean number of IED per session across all patients and sessions was 112.4 (standard deviation 88). Patients had between 1 and 4 types of IED with 55% of patients having 1 type, 29% having two types and 13% having 3. Only one patient had 4 different types of IED.Using an ANOVA it was found that there was no statistically significant effect of condition  on IED rate, F = 0.273, p>0.05 .2  = 0.81, p>0.05)There was no significant effect of movie stimuli on the concordance of EEG-fMRI maps: the proportion of concordant maps derived from sessions with the movie stimuli did not significantly differ from the proportion of concordant maps derived from resting sessions (χThe EEG-fMRI maps for 18 out of 28 (64%) patients who completed the 4 sessions were classified as concordant. When considering the first 20 minutes of scanning alone (2 sessions), the proportion of concordant maps was very similar 63% (24/38). This was despite the potential increase in age and compliance of the group who could tolerate 4 sessions and the increase in available data.The classification accuracy of the logistic regression of design efficiency on EEG-fMRI concordance was 65%. This was found to be a statistically significant effect .The classification accuracy of the logistic regression model investigating the factors that predict concordance had a classification accuracy of 65%. The effect of motion, as measured by mean framewise displacement, was found not to be statistically significant . TSNR was did not predict concordance either . However, design efficiency was a statistically significant predictor of concordance .EEG-fMRI studies in epilepsy have been much more frequently performed in adult patients compared to children. The greater challenges of scanning children may in part explain this difference. However the potential advantages of well targeted surgical intervention in children make it important to develop strategies that facilitate brain imaging techniques in this population. In addition, tolerable and minimally invasive tests with reduced health risks (i.e. those associated with ionising radiation or anaesthesia) are particularly important in children.In this study, a more child-friendly approach to EEG-fMRI was implemented and assessed in children with epilepsy in the age range 6–18 without using sedation which is one of the largest cohorts of children whom have undergone EEG-fMRI scans. To our knowledge, this is the first EEG-fMRI study in epilepsy using stimulation during the recording.Sedation has been the approach of choice in paediatric EEG-fMRI studies . SpecifiWe recorded a high rate of tolerance of the protocol among our population and we achieved longer scanning durations compared to the average resting state (5–10 minutes) , 36 and EEG-fMRI is typically recorded in resting state with eyes closed  under thIn addition to showing that there was no penalty for increasing the tolerability of EEG-fMRI for children this opens the door to the possibility that EEG-fMRI studies during stimulus could be effective for localisation of focal epilepsy in tandem with cognitive fMRI that is frequently obtained for motor and language mapping  in this We demonstrated that design efficiency is predictive of EEG-fMRI concordance, this suggest that not only the rate but the combination of rate and the distribution in which IED occur is important for finding significant results. To our knowledge no EEG-fMRI study of patients with epilepsy has examined the effect of design efficiency on the quality of the EEG-fMRI result. Design efficiency of the EEG-fMRI experiment provides a measure of the power of the test to map the epileptogenic region, therefore it is recommended that EEG-fMRI studies in patients with epilepsy measure their design efficiencies and interpret and report their results in light of them.The results presented here represent the first systematic attempt to address the optimal scan duration. Typical scan times in adult EEG-fMRI studies range from 40 to 90 minutes in order to maximize the chances of capturing IED inside the scanner , 9. WhilWe acknowledge several limitations to this study.Firstly, our results may be limited by the accuracy of the measure chosen to classify concordance of EEG-fMRI maps. The epileptogenic region was defined by the presurgical tests as opposed to the surgical outcome which is considered the gold standard to define this region. Therefore it was not possible to assess the true positive or negative predictive value of the EEG-fMRI results. The concordance measure used however provides a metric that allowed the objective comparison of the different factors that contribute to the ability of EEG-fMRI to map IEDs.Secondly, EEG-fMRI studies in epilepsy may be limited by the hemodynamic response models used. While there is not a consensus about the best hemodynamic response model to use in these studies , the canLastly, the conclusions about the optimal duration of EEG-fMRI can be extrapolated to a subpopulation of patients with frequent spikes such as the one recruited in this study. In subjects with infrequent activity longer scanning times may be optimal.In conclusion our data shows that good quality EEG-fMRI data can be obtained in children as small as six without sedation. Child friendly stimulus such as movies improve the tolerability of the test and reduce the movement inside the scanner without having a significant effect on the epileptic activity and/or the EEG-fMRI maps.Twenty minutes of EEG-fMRI acquisition seem to be an optimal duration that maximizes the likelihood for obtaining significant results and to minimize in-scanner movement in our patient group with frequent IEDs.Design efficacy derived from the occurrence of interictal epileptiform discharges is a relevant factor in the quality of EEG-fMRI maps, the quantification of this factor may help in the interpretation of EEG-fMRI results.S1 FigFive examples of individual movement parameters across the different scan sessions. Blue, red and green lines represent the rotation (y axes) on the three axes in space across time/images (x axes) relative to the first image. Each session comprises 300 images, different sessions are separated by vertical dotted lines. For each case the type of session (video/ rest) is represented underneath the X axes by Tom & Jerry picture (movie session) and eyes closes (rest session). A and B are representative of the most common pattern across subjects showing increased movement after 2 sessions and an effect of video in the first/second session. D and E show two examples with large effect of video on the movement. It is even noted increase of movement during the “please wait” screen. C represents a patient who fell sleep after the first session with video.(TIFF)Click here for additional data file.


Electrical stimulation of the lateral hypothalamus can motivate feeding or can serve as a reward in its own right. It remains unclear whether the same or independent but anatomically overlapping circuitries mediate the two effects. Electrical stimulation findings implicate medial forebrain bundle (MFB) fibers of passage in both effects, and optogenetic studies confirm a contribution from fibers originating in the lateral hypothalamic area and projecting to or through the ventral tegmental area. Here we report that optogenetic activation of ventral tegmental fibers from cells of origin in more anterior or posterior portions of the MFB failed to induce either reward or feeding. The feeding and reward induced by optogenetic activation of fibers from the lateral hypothalamic cells of origin were influenced similarly by variations in stimulation pulse width and pulse frequency, consistent with the hypothesis of a common substrate for the two effects. There were, however, several cases where feeding but not self-stimulation or self-stimulation but not feeding were induced, consistent with the hypothesis that distinct but anatomically overlapping systems mediate the two effects. Thus while optogenetic stimulation provides a more selective tool for characterizing the mechanisms of stimulation-induced feeding and reward, it does not yet resolve the question of common or independent substrates. Electrical stimulation of the same sites in the lateral hypothalamic area (LHA) can cause a drive-like state—inducing feeding in sated animals—or it can serve as a reward in its own right ,2. That The LHA was initially identified as a feeding center by some workers ,12, and The intrinsic neurons of the LHA form a segment the interstitial or “bed” nuclei feeding efferent fibers into the (MFB) ,20. SeveParametric electrical stimulation studies have confirmed that fibers linking the LH and VTA are involved in both the rewarding  and the Optogenetic stimulation offers a way to selectively activate fibers originating from more or less localized cell populations . Such stMale Sprague-Dawley rats  weighing 275-350g were used. They were individually housed in a temperature and humidity-controlled room with a reverse 12-h dark-light cycle (lights off at 0800h) with free access to dry food and water. Animal procedures were performed in accordance to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and were approved by the Animal Care and Use Committee of the National Institute on Drug Abuse Intramural Research Program.2), each rat was injected with a viral vector targeting one of the medial forebrain bundle sites of interest and a fiber optics probe just dorsal to the ventral tegmental area. The tissue over the skull was resected, and subcutaneous injections of the local anesthetic Marcain (0.1 ml of a 0.5% solution) were given at the wound margins. Expression of the light sensitive cation channel channelrhodopsin-2 (CHr2) and the marker enhanced yellow flourescent protein) were induced by unilateral injection of 0.5μl the viral construct rAAV5-CamKII-hChR2(H134R)-eYFP (University of North Carolina Vector Core) at a rate of 0.1μl/min. The four injection targets, separated by 1.0 mm in the anterior-posterior plane, were the lateral preoptic area (LPO: AP -0.8), anterior hypothalamic area (AHA: AP -1.8), lateral hypothalamic area (LHA: AP -2.8), and posterior hypothalamic area (PHA: AP-3.8); the medial lateral (ML 1.7) and dorsal ventral (DV -8.4) coordinates were common to the four sites (coordinates according to [Under isoflurane anesthesia (2–3% isoflurane in 1L/min Ording to ). A grouEach rat was tested for stimulation-induced feeding and self-stimulation under dim red illumination in a standard operant chamber (Med Associates) equipped with two nose-poke ports with white cue lights above the ports. During testing the rats were connected to a 200μm fiber optic cable (Thorlabs) through a rotary optic joint (Doric Lenses) to a laser (OEM Laser Systems) used to deliver 473nm blue light under the control of a digital pulse simulator .Each animal was tested for stimulation-induced feeding and for self-stimulation; half the animals were tested for feeding first and half for self-stimulation first. Initial screening for stimulation-induced feeding involved 10 one-minute trials with continuous 20Hz trains of 5msec laser pulses interspersed with 10 one-minute unstimulated comparison trials. Approximately 40g of dry lab chow was scattered on the floor of the test chamber and latency to pick up and eat food was scored in each stimulation or no-stimulation trial. Eating was identified as stimulation-induced if three criteria were met: (i) the animal initiated eating in 8 of the 10 daily stimulation periods on each of 5 consecutive days of testing, (ii) once eating was initiated within a trial it continued until the stimulation terminated at the end of the trial, and (iii) eating was never initiated in the one-minute no-stimulation trials. Latencies to first bite and amounts eaten were each recorded.Twelve rats that both ate and self-stimulated in response to 20Hz stimulation were tested further under several variations in stimulation parameters. Four stimulation frequencies  were each tested with each of three pulse widths ; stimulation parameters were tested on separate days and given in a counterbalanced sequence. The 8 animals infected with eYFP without ChR2 were tested with this group and in the parametric testing for self-stimulation.Each rat was independently trained and tested in a nose-poke apparatus to determine if optical stimulation was rewarding. In initial screening, each interruption of a photocell beam in one of two ports (the “active” port) triggered a 1.5sec train of 5ms pulses of optical stimulation at 20 Hz. Each nose poke also activated a white cue light above the active port. Responding during a stimulation train or in the other (“inactive”) port was without scheduled consequence. Rats were trained in daily 10-min sessions until the rate of beam interruptions varied less than 10% over 3 consecutive days. Animals that failed to perform at least 20 active nose-poke responses per session were considered as non-responders and were not used further. Rats that displayed stable response rates were tested in a reversal task in which nose-poking in the previously active port was no longer effective but poking in the previously inactive port became effective.The twelve rats that were tested for feeding under parametric variations were tested for self-stimulation with the same stimulation frequencies  and the same three pulse widths  and with each of six train durations ; each combination was tested on a separate day and the combinations were tested in counterbalanced sequence.2PO4 in 1L of distilled water) following by a 4% paraformaldehyde solution. Brains were removed, left in 4% paraformaldehyde for 2 hours at 4°C and dehydrated overnight in a phosphate buffer solution containing 18% sucrose at 4°C. The next day, the brains were transferred to a fresh phosphate buffer solution containing 18% sucrose at 4°C for 3 hours before being frozen and stored at -80°C. Sagittal or coronal sections were subsequently taken at 20 um and stored at -80°C in 0.1% DEPC-treated water solution followed by 30% sucrose, 30% ethylene glycol and 2mM of NaN3.Following completion of testing the rats were anesthetized with equithesin  or isoflurane and were transcardically perfused with a phosphate buffer solution .Brain sections were processed for eYFP-immunodetection as follows: free-floating coronal sections were incubated for 1 h in PB supplemented with 4% bovine serum albumin and 0.3% Triton X-100. Sections were then incubated with mouse anti-GFP antibody  overnight at 4°C. After rinsing 3 × 10 min in PB and incubated in biotinylated goat anti-mouse antibody ; then, sections were rinsed with PB and incubated for 1 h at room temperature in avidin-biotinylated horseradish peroxidase . Sections were rinsed, and the peroxidase reaction was developed with 0.05% 3,3’-diaminobenzidine and 0.03% HThe effects of pulse width and pulse frequency were assessed in the feeding study with 2-way and in the self-stimulation study with 3-way within-subjects analysis of variance (ANOVA). Post hoc comparisons were made using Bonferroni correction. When the assumption of sphericity was not met, conservative Greenhouse-Geisser corrections were made to the degrees of freedom. Separate within-subjects analyses were done for train lengths of 200-800msec and 1600-6400msec because independent groups were used for the two ranges. Linear and quadratic trends were assessed with 1-way ANOVAs for each frequency in the feeding study. Calculations were performed using SPSS software (IBM Corporation).Optical stimulation of the VTA induced feeding and was rewarding in some but not all animals with viral transfection of ChR2 plus eYFP into cells of origin at various levels of the hypothalamic MFB. The effectiveness of stimulation varied with the site of the viral injections, as discussed in the following section. Optical stimulation of the VTA never induced feeding and was never rewarding in any animal transfected with eYFP but not ChR2.Viral infections spread approximately 1mm from each injection site; thus the infections at each site overlapped considerably with those of neighboring sites . Optic p2,24 = 10.447, P<0.001 and F1.661,19.934 = 49.936, P<0.001), amounts eaten , and self-stimulation rate , there were in each case significant Pulse Width X Pulse Frequency interactions . The interactions reflected the fact that whereas the effects of stimulation frequency were predominantly linear in the case of short pulse duration, they were markedly non-linear in the case long pulse duration. This is illustrated in the case of amounts eaten by a strong linear and a weak quadratic trend for the 2.5ms pulse width  and a strong quadratic trend and an insignificant linear trend  for the 10m pulse width . In the case of each of the eight testing conditions, the 2.5ms pulses were more effective at 80Hz than at 40Hz  whereas in each of the eight conditions the 10ms pulses were less effective at 80Hz than at 40Hz (P<0.02). In each case 5m pulses at 80Hz was intermediate to the strong effect of 2.5ms pulses and the weak effect of 80Hz pulses .The effectiveness of stimulation varied as complex functions of pulse width and pulse frequency in the case of feeding  and of pP<0.001 in each case).The most effective frequency for stimulation-induced eating was 80Hz with 2.5ms pulses and 40Hz with 5 or 10ms pulses. The most effective frequency for self-stimulation was 80Hz for 2.5ms pulses, 80Hz for short trains and 40Hz for long trains of 5ms pulses, 40Hz for long trains and all but the shortest train of 10ms pulses . AmountsAnimals with LHA viral injections that ate in response to 20Hz stimulation sometimes picked up cardboard pieces when tested with food and cardboard present; on each such occasion, however, they simply moved the cardboard aside and picked up and began eating a food pellet. In the nine animals that ate in response to stimulation and were subsequently tested with water rather than food, no instances of stimulation-induced drinking were seen at any of the combinations of parameters previously used in the feeding tests.At the stimulation parameters used—and as it the case with electrical stimulation —the feedThe present findings suggest a substrate for both feeding and reward that originates in cells of the lateral hypothalamic area at the anterior-posterior level of the ventromedial nucleus—the area classically associated with feeding—projects to or through the ventral tegmental area and does not involve fibers from more rostral or caudal bed nuclei of the MFB. The fact that the anterior-posterior level of the lateral hypothalamus is identified with respect to the adjacent ventromedial nucleus reflects the fact that the lateral hypothalamic area is merely a portion of ascending columns of glutamatergic and GABAergic neurons that form bed nuclei of the medial forebrain bundle. There are no clear boundaries of this region with respect to the cells infected by our viral injections, and the considerable overlap of our injections does not provide such boundaries. Nonetheless, neither eating nor self-stimulation was seen in cases that were not in or adjacent to the LHA, and only fragmentary findings were seen in cases not targeting the LHA. The findings, while consistent with the view that a common substrate mediates the two findings, do not falsify the alternative and do not resolve the question of common or separate substrates. Whereas the region cannot be delimited by the boundaries of glutamatergic or GABAergic nuclei, several more punctate cell groups can be identified by co-expressed neuropeptides. It is possible that the targeting of ChR2 to selected neuropeptide-expressing populations will allow a precise delimitation of the critical substrates for feeding and reward in this region; such studies will offer further opportunities to dissociated the mechanisms of the two behaviors.A common system is suggested by the findings that both responses were seen in the majority of animals that showed either response but a common system is questioned by the findings that 11 animals ate but did not self-stimulate and seven that self-stimulated but did not eat. All the animals that ate or self-stimulated were in the lateral hypothalamic group or one of the adjacent groups with viral infections that overlapped the lateral hypothalamus. If the eaters that did not self-stimulate had all been from one group and the self-stimulators that did not eat from another group, this would have been strong evidence for separate but overlapping systems. This, however, was not the case. Animals that ate but did not self-stimulate were distributed across the AHA, LHA, and PHA groups, and animals that self-stimulated but did not eat were distributed across both the AHA and PHA groups. Our working hypothesis is that the behaviors in the AHA and PHA animals were less reliable simply because their infections only partially invaded the critical lateral hypothalamic substrate or substrates. None of the lateral preoptic animals, whose infections overlapped those of the anterior but not the lateral hypothalamic groups showed any inclination to eat or self-stimulate.It is difficult to understand the finding that nine of the 11 animals that ate but did not self-stimulate were within the group with LHA viral injections. Given the size of the infections, it is difficult to imagine that small differences in the stereotaxic accuracy of the viral injections could account for the lack of self-stimulation in these animals; small differences in electrode placement do not cause significant differences in feeding or self-stimulation induced by electrical stimulation ,43. SmalThe parametric findings are consistent with findings from electrical stimulation studies ,6 suggesThe viral approach used in the present study established non-selective optogenetic control of both GABAergic and glutamatergic projections from the LHA to the VTA . It has As expected from electrical stimulation studies, response rates in the present study were inversely related to train duration. In large part, this reflects the fact that longer stimulation trains take up a greater portion of the test session and require fewer responses to maintain the same rate of stimulation. Another possible factor, however, is that stimulation that is initially rewarding often becomes aversive ,46 or atWhile the finding that activation of GABAergic but not glutamatergic fibers from the LHA to the VTA support feeding and self-stimulation suggests a common GABAergic substrate for the two behaviors, it does not rule out the possibility that different subsets of GABAergic fibers are responsible for the two effects. Calcium imaging and electrophysiological recording studies indicate subpopulations of LHA neurons, at least some of which project to VTA, that are involved in different aspects of feeding and food-approach. Thus it remains to be determined whether the same population of GABAergic LHA to VTA neurons mediates stimulation-induced eating and self-stimulation.S1 File(XLSX)Click here for additional data file.


Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest such atlas yet assembled. We found that the early transcriptional response mirrors the distinctiveness of physiological origins: genes respond in restricted patterns, conditional on the expression levels of dozens of transcription factors. Only a small cohort of genes is constitutively modulated independent of initial cell state. Ecdysone-responsive genes tend to organize into directional same-stranded units, with consecutive genes induced from the same strand. Here, we identify half of the ecdysone receptor heterodimer as the primary rate-limiting step in the response, and find that initial receptor isoform levels modulate the activated cohort of target transcription factors. This atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly.Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone . In the case of 20-hydroxyecdysone (ecdysone) in  Among the most striking of these developmental effects is the coordination of insect development and metamorphosis by the steroid molting hormone 20-hydroxyecdysone . At metamorphosis, virtually every cell responds to the hormone, but the responses are as diverse as the adult organism that is being formed: many cells that played important roles during larval life are stimulated to start down pathways leading to programmed cell death, while others, destined to form adult tissues, enter pathways leading to differentiation and morphogenesis , which are deeply conserved across metazoans. These NHRs share a common structure: an unconserved N-terminal A/B region including a transcriptional activation domain (AF-1), followed by a highly conserved 66-68 residue DNA binding domain (DBD), a short hinge region, a conserved ligand binding domain (LBD), and unconserved sequences of variable extent called the F domain. Receptors function by binding to highly conserved response elements (HREs), where they act as powerful transcriptional effectors.EcR and ultraspiracle (usp). EcR binds hormone only as part of the heterodimer (Ecdysone acts through a heterodimeric NHR composed of the products of the genes erodimer . The “caerodimer . EcREs aDrosophila ATF4) is known to interact directly with isoform B2  is known to be crucial to ligand-independent repression  a limited number of direct coding and noncoding target genes including transcription factors (TFs) and microRNAs . These rll lines , 1994. All lines , inductiThese observations frame a central question: In any one cell, at any one stage, how are responding genes selected from the broad array of potential targets? Few genome-wide studies have been conducted of the ecdysone response. Following initial work using subsets of genes and microarrays , and grown as recommended there. Each cell line used in this paper has a formal name, which is the unique identifier used by FlyBase and the DGRC. For convenience, we use a shortened version of many of these cell line names in this paper; both the formal names and the short names are given in The individual cell lines are listed in All of the cell lines were of independent origin. S2 cells have been grown for many years in a large number of laboratories, under variable conditions, and different isolates from the same original line  often ha−6 M final concentration, Sigma-Aldrich) as described previously . Statistically significant responsive genes are those that achieved either an adjusted p-value less than 0.01 regardless of Fisher’s Method p-value, or an unadjusted p-value of 0.01 and a Fisher’s Method p-value less than 10http://homer.salk.edu/homer/motif/.Enrichment of motifs within promoter region DNA sequences was carried out using the homer2 program  using custom scripts. Gene lengths for length-normalized expression were taken as the mean of the lengths of the transcripts for that gene. GO term enrichment was produced using the fb_2014_03 version of the FlyBase gene ontology . The selected transcription start sites are included in Table S5.For all significantly responsive genes, the fraction of responsive genes and the average response direction of nearby genes were analyzed. In order to determine the distance between two genes, the ecdysone relevant transcription start sites (TSS) were first determined. For genes with multiple transcription start sites, the TSS was determined to be the TSS associated with a significant exon level response to ecdysone if one exists, or the exon with the highest length normalized expression in the relevant time point . A moving window of 100 gene–cell line combinations was used to calculate the fraction of responsive genes and the average response direction within each bin. The binned points were grouped according to the response direction of the responsive gene as well as the shared promoter architecture (upstream/downstream and same/opposite strand). These points were then smoothed using LOESS with a local linear fit over binned points. The plot produced is found in In order to predict the restricted response, the following two models (referred to as matched induction and matched repression) were fit. The models are symmetric, so only the matched induced model will be described in full detail.At each gene in the restricted induced set, we aimed to predict which cell lines responded significantly and which responded most repressively, defined as the cell lines showing the smallest log2 fold change of ecdysone exposure expression/preexposure expression. A matching number of the most repressive responsive cell lines were chosen such that each gene contained a balanced number of induced and matched repressed cell lines.In order to predict which gene–cell line combinations belonged to the above described sets, the model was provided with normalized TF expression masked by a known TF motif from the TOMTOM database  that have an extended time course, including the 0, 1, 3, 5, and 7 hr time points, the following analysis pipeline was conducted. In order to compare the transcriptional responses across the time course for each cell line, a normalization was performed that allows comparison of genes with dissimilar steady state expression levels, but may share ecdysone response “shape.” This normalization begins by applying the robust median library size normalization from the DESeq R package . Then, aTable S2 Micraarray data re in the GEO database at NCBI as series GSE11167.All sequences are available through the SRA (BioProject PRJNA306537) and individual sample accession numbers are available in Table S1) before and after a 5 hr exposure to ecdysone at a biologically relevant concentration of 10−6 M. Transcription levels were measured by single-end poly(A)+ RNA-sequencing with 100 bp reads. For four of the cell lines, CCa, Kc, MCW12 and BG3-c2, duplicate samples were collected in order to estimate the biological variation present in this system. Samples were taken from an extended time course of exposure at 1, 3, 5, and 7 hr for three cell lines, Kc, BG3-c2 and S2-DRSC. All sequences are available through the SRA (BioProject PRJNA306537) and individual sample accession numbers are available in Table S2. Gene and exon-level transcriptional quantifications were assessed using the FlyBase r5.57 annotation (Table S3 and Table S4). Differential expression calculations were carried out using the DESeq . Significantly induced or repressed genes were identified by applying a biological relevance fold change threshold of two and a statistical significance threshold of 0.01 adjusted p-value. A relaxed statistical significance threshold of 0.01 unadjusted p-value was applied for genes that showed a strongly significant tendency for induction or repression across many cell lines . Differentially expressed (DE) genes along with meta-information can be found in Table S5 and Table S6. RNA samples were collected from 41 cell lines  using DGRC-2 microarrays . Effects were scored as significant at an FDR threshold of 1%. All results have been deposited in GEO. Data were biologically replicated in triplicate and results can be found in Drosophila genes  are expressed at greater than one RPKM (reads per kb per million mapped reads) in at least one of the 41 cell lines we profiled at ground state (prior to ecdysone exposure). Of those, 5846 are constitutively expressed in all cell lines, while 1459 are expressed in only a single cell line. The number of expressed TFs per cell line ranges from 406 to 450, and 595 . After ecdysone exposure, an additional 305 genes are expressed that were not basally detectable in any cell line and, conversely, 360 genes are constitutively inactivated after ecdysone exposure. We defined genes with significant responses in more than half of the cell lines as “widespread,” and others as “restricted.”Cell lines are remarkably transcriptionally diverse . The two most striking clusters of cell lines . An additional activated Ras line (Ras-H7) shows some similarity in its responses. If confirmed, the existence of a Ras/Pten cluster could suggest that the pathway to generation of a cell line by either gain of Ras function or loss of Pten function leads to a transcriptional environment conducive to a particular pattern of ecdysone response.We note that the collection of cell lines includes six created by expression of a constitutively active Ras oncogene , a null mutation in the tumor suppressor gene Pten (PtenX), or both (PR-8) (Table S5). Several of these are known ecdysone-responsive TFs, while many are newly identified and point to new hormone-responsive pathways. Table S7 provides enriched GO terms among induced and repressed genes.A total of 1645 genes are significantly transcriptionally responsive in at least one cell line. Fifty-nine TFs are induced in response to ecdysone, and 35 of these are responsive in five or fewer cell lines . The RGC varies by two orders of magnitude across cell lines and is driven by responsive genes with highly restricted expression patterns .It is well known that some tissues and cell types are more responsive to ecdysone than others. We measured the responsiveness of a cell line as the count of genes significantly induced or repressed 5 hr after induction, and refer to this as the Responsive Gene Count (RGC). While RGC is threshold-dependent, the rank-order of cellular responsiveness is well preserved across a broad range of biologically and statistically meaningful parameterizations . Among all genes, the expression level of EcR is by far the most strongly correlated with RGC . We further assessed this observation within both induced and repressed genes . The EcR heterodimer partner, usp, exhibits weak correlation with RGC  and correlates only weakly (and inversely) with the total number of genes expressed per cell line . We assessed the association of the RGC with the basal expression level of each gene in the genome in a multiple testing setting , show little correlation with any gene . Taken together, these results indicate that EcR titer is rate-limiting for the transcriptional response to ecdysone.The residuals left after correcting for the effect of Hr4, Hormone receptor-like in 46 (Hr46), Ecdysone-induced protein 75B (Eip75B), CG44004, and bip1, are induced in all 41 cell lines. All five have been previously identified in other genome-wide surveys of ecdysone exposure response (Table S10). There are no genes repressed in all cell lines, with fruitless (fru) being the most widespread (repressed in 33 of 41 cell lines). More broadly, widespread induced genes are significantly enriched for biological GO terms “metamorphosis,” “salivary gland cell autophagic cell death” and “steroid hormone mediated signaling” , as expected, since the majority of these genes have been previously reported in other studies of ecdysone response (Table S10).The 100 genes with widespread responses include 68 induced and 32 repressed in more than half of our cell lines. Five genes, Materials and Methods).The promoter regions of widespread induced genes, defined as 500 bp upstream of each TSS, are 36% enriched over background for the EcRE motif , representing the most significantly enriched of all known motifs in the HOMER library  in the 4–14 hr embryos, consistent with response to the midembryological ecdysone pulse. This is in contrast to the majority of lncRNAs in Drosophila , which are expressed predominantly in tissues of the nervous system and the gonads  peptides critical for ecdysone transduction in the epidermis  genes, The set of widespread repressed genes is much smaller than the set of induced genes, as is the overall repressive response in most cell lines. There are no statistically enriched GO terms among this set of genes.Materials and Methods).We find that 93% (863) of induced genes and 96% (756) of repressed genes are affected by ecdysone in fewer than half our cell lines. These restricted responses form the molecular basis of the diverse transcriptional and physiological effects that the hormone induces throughout development and within distinct cell types. A total of 400 and 241 genes are induced and repressed, respectively, in exactly one cell line, with a large fraction (31%) responding only in the Ras-H3 cell line, an outlier in this study (see Table S11). Sixteen are induced and repressed in at least two cell lines. One striking example is the TF CG9932, which is significantly induced in five cell lines and significantly repressed in six. CG9932 is differentially expressed across development with peaks at 20 hr and late L3 stage and shows strong expression in embryonic fat body and salivary glands. It is likely that some promoters respond in distinct directions based on prior epigenetic state. This phenomenon has also been noted in mammalian response to glucocorticoids . A total of 635 genes, 335 induced and 300 repressed, of this type are present genome-wide. Hence, leveraging our large number of cell lines we are able to confidently detect weak modulation of a large cohort of genes. These relatively small perturbations may be of no physiological significance to the cell; however, this may not always be the case. Weakly induced genes are strongly enriched for GO terms including “protein binding,” “vesicle-mediated transport,” and “macroautophagy,” and weakly repressed genes are enriched for “rRNA processing” and “calmodulin binding” (Table S7). Indeed, all five core TFIIH complex genes  are weakly repressed in at least two-thirds of cell lines, while not a single gene is significantly responsive. While each of these individual modulations is small, the cumulative effect in a given cell on the titer of the functional TFIIH complex may be substantial. Genome-wide, weakly induced genes across most cell lines are found within 2.5 kb of a significantly induced gene more often than expected by chance . This pattern does not hold for weakly repressed genes.The restricted set includes genes that respond weakly in several cell lines. Some of these genes do not pass our statistical criteria in any single sample, but by aggregating information across cell lines we obtain sufficient power to confidently annotate weak, reproducible induction or repression  and additionally tend to respond in the same direction . The response of opposite-strand gene pairs  is less coordinated than genes with operon-type architecture . One striking example of a divergently responsive bidirectional pair is Piezo and CG8498. The TSSs of these genes are separated by only 655 bp , a protein involved in chromatin remodeling . The invEip75B shows the strongest exon level event in the genome across all cell lines , consistent with previous reports  play important roles in development. EcR isoforms are differentially expressed between Kc cells and salivary gland cells, indicating that alternate EcR isoforms elicit different transcriptional responses. Cell lines in this study show a wide range of EcR isoform expression. The length normalized fraction of the EcR-B1/2 isoform expression  ranges from 0.31 (BG3-c2 cell line) to 1 (S2 cell line) with most cell lines expressing predominantly the EcR-B1/2 isoform. We do not see strong correlation to the total number of induced or repressed genes, or the residuals after correcting for the main EcR expression effect, but we do observe strong correlation between the EcR-B1/2 isoform fraction and the expression of many individual genes. The most significantly correlated genes are gliolectin (glec), squeeze (sqz), CG5059, Eip55E, and broad (br) (top 100 genes are listed in Table S13). Only glec and Xbp1 show increased expression with increased EcR-A isoform levels among the top 10 most correlated, consistent with a predominantly repressive role for EcR-A . Of the highly correlated genes, the TF br shows the largest dynamic range (two orders of magnitude). Twenty-three cell lines show significant induction and one, BG3-c2, shows significant repression  is well characterized by EcR titer, other effects are clearly at work producing diverse restricted responses. TFs are likely candidate EcR cooperative factors. We developed a statistical machine-learning model aimed at identifying these factors: we used basal expression levels and TF binding motifs to predict restricted responses. We also supplied the model with information about the basal expression level of each gene, since it is more difficult to detect repression of genes that are poorly expressed, and to detect induction of genes that are highly expressed.We fit models for induction and repression. A notable feature of our modeling strategy is that we hold out entire cell lines, so when we assess our model’s predictive performance, it is assessed on cell lines it has not previously seen. This ensures that our model is generalizable to unseen cell lines. We find a predictive accuracy on held-out cell lines of 61% and 64% for induced and repressed genes, respectively, indicating that we have weak but significant power to predict the direction of a gene’s response.Materials and Methods). We find that, for models of both induction and repression, the rank of basal gene expression level is the most important covariate. For the repression model, we see that gene rank is two and half times more important than the most important TF. In the induction model, the gene rank is only one and one-third times more important. However, in both models, several TFs also show comparably significant importance values (listed in Table S14). The most important TFs include known ecdysone response factors br and Eip74EF. TFs not previously implicated in the ecdysone response are also important; these include longitudinals lacking (lola) and Chorion factor 2 (Cf2). We note nearly all covariates have weak positive values, indicating that the restricted response is a function of a large number of TFs, and hence that ecdysone response depends in detail on the expressed cohort of TFs.We used feature selection to compute the relative importance of each covariate in our model . Therefore, transcriptional profiling alone is not sufficient to fully elucidate the ecdysone response.This approach also enables us to assess the power of transcriptional profiling for elucidating the basis of hormone responses. We fitted and assessed this model successively using different numbers of cell lines, between 4 and 40 . From this analysis, we extrapolated a theoretical maximal accuracy (asymptote) for identifying the response of an unseen cell line of 74% and 79%, respectively, given an infinite number of cell lines in the training set . This intuitive representation captures much of the same information as z-scores, and has the advantage that each gene is set to the same (zero) value in basal conditions. We used this representation to identify structures in both the scales and directionalities of temporal responses , including br and Eip71CD. The rapid (at 1 hr) induction of these genes indicates that BG3-c2 is in a more ecdysone sensitive basal state. Notably, the total responsiveness of these three cell lines differs substantially: the RGC value for BG3-c2 cells is more than twice that of S2 or Kc cells. Taken together, these data indicate that the observed increased responsiveness of BG3-c2 cells at 5 hr may be due to an accelerated ecdysone response relative to other cells.There are 15 genes that are responsive at 5 hr after induction only in Kc and S2 cells but not in BG3-c2, and these show a strong and consistent expression pattern in the BG3-c2 time course: they are responsive at 1 hr, and then reduce expression level at each time point thereafter. Six of these 15 genes are widespread responsive  varies among the panel of lines. These genes are of special interest because understanding how their behaviors are dictated by cellular state encapsulates the problem we are investigating.tou supports the idea that the secondary targets may be determined in part by chromatin remodeling, and that cofactors, in addition to TFs, play essential roles in specifying cellular responses.The organization of responsive genes in the genome supports the idea that epigenetic state, including chromatin context, is prominent. Responsive proximal gene pairs preferentially organize on the same strand and a number of bidirectional promoters are found to be under independent control. This is remarkable and indicates that the spatial resolution of ecdysone transduction along the genome is on the order of hundreds of base pairs. Additionally, this responsive gene architecture suggests a role for Pol2-associated chromatin modifications. Furthermore, the widespread and early induction of genes encoding chromatin remodeling factors like br and lola are strong predictors of restricted responses across all 41 cell lines. We estimate that more than a hundred TFs are needed to achieve maximal predictive accuracy, indicating that the interaction of many TFs and cofactors convey divergent gene responses as previously reported  to a promoter or enhancer sequence that evolved for other purposes. Or, alternatively, as is indicated by the coresponsiveness of proximal genes transcribed on the same strand, local chromatin modifications needed to active a target gene may have auxiliary effects in the genomic neighborhood. A response that is physiologically unnecessary but not harmful will be maintained if the components of the response are needed for other purposes, e.g., in other cell types. Whatever the biological significance (or lack of it) for downstream cytological events of these minor expression changes, understanding the regulatory events that cause them can be expected to shed light on the regulatory syntax we are studying.We count ecdysone-responsive genes by looking for in vivo Drosophila cells. They respond to a common signal with diverse responses that must be specified by differing initial states. More detailed studies of these responses, expanded to include small RNAs and coupled with binding site maps for relevant transcription factors, chromatin state profiling, and judiciously designed perturbations, should make it possible to increase the accuracy of prediction and elucidate the underlying biology of hormone response in metazoans.The catalog we have produced, along with our initial successes in predicting ecdysone responses, indicate the potential of the system as a model for hormone response. In our view these 41 cell lines, representing 41 individual systems, are governed by regulatory rules that are likely identical, or nearly so, to those of


TaGA20ox1 homoeologous genes. After testing different mapping algorithms and settings, candidate SNPs were identified by mapping to the IWGSC wheat Chromosome Survey Sequences. Where sequence data for all three homoeologues were found in the reference, mutant calls were unambiguous; however, where the reference lacked one or two of the homoeologues, captured reads from these genes were mis-mapped to other homoeologues, resulting either in dilution of the variant allele frequency or assignment of mutations to the wrong homoeologue. Competitive PCR assays were used to validate the putative SNPs and estimate cut-off levels for SNP filtering. At least 464 high-confidence SNPs were detected across the three mutagenized lines, including the three known alleles in TaGA20ox1, indicating a mutation rate of ~35 SNPs per Mb, similar to that estimated by PCR-based TILLING. This demonstrates the feasibility of using exon capture for genome re-sequencing as a method of mutation detection in polyploid wheat, but accurate mutation calling will require an improved genomic reference with more comprehensive coverage of homoeologues.Targeted Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach to identify novel sequence variation in genomes, with the aims of investigating gene function and/or developing useful alleles for breeding. Despite recent advances in wheat genomics, most current TILLING methods are low to medium in throughput, being based on PCR amplification of the target genes. We performed a pilot-scale evaluation of TILLING in wheat by next-generation sequencing through exon capture. An oligonucleotide-based enrichment array covering ~2 Mbp of wheat coding sequence was used to carry out exon capture and sequencing on three mutagenised lines of wheat containing previously-identified mutations in the  The approach has been particularly successful in diploid species such as rice, in which mutations affecting gene function are more likely to have a detectable phenotype. However, many important crop species are polyploid, and although the genetic buffering afforded by the multiple gene copies permits a higher rate of mutation) 5 generation and wheat is largely self-fertilised, 88% of the EMS mutations would be expected to be homozygous  with 12% heterozygous . However, mapping to all three reference genomes and filtering for SNPs with a minimum of 3 supporting reads resulted in the detection of very significant numbers of SNPs with allele frequencies below 0.4  the proportion of EMS-type SNPs is 97%, suggesting that nearly all are correctly called, while even at low allele frequencies (≤0.4) the proportion of EMS-type SNPs was 43%, higher than would be expected by chance. This suggests that a significant number of SNPs detected at allele frequencies as low as 0.2 could be valid EMS mutations, although the rate of false positives is expected to be high. However, As the material used for this study was from EMS-mutagenized plants at the M5 lines being 88% homozygous, is that the IWGSC Chromosome arm Survey Sequence[One possible explanation for the high proportion of candidate mutations with low observed allele frequencies, despite the mutations in the M Sequence used as TaGA20ox1 gene  in each of which a confirmed EMS mutation in the A homoeologue was known. Results from mapping reads from the three mutant lines, C6, A6 and D3, to the TaGA20ox1 genes in these modified reference genomes are shown in TaGA20ox1 homoeologues in the reference resulted in the detection of the known mutations at an allele frequency of 1.0 (i.e. homozygous), but the removal of one or more homoeologues from the reference in most cases resulted in a decrease in the allele frequency of the mutant SNPs due to mis-mapping of reads from other homoeologues. This is illustrated in TaGA20ox-A1 in line C6: the variant reads corresponding to the G>A mutation at position 1018 are diluted by wild type reads, particularly from the D homoeologue, when the reference is incomplete. Thus in the presence of all three homoeologues in the reference, the mutation is correctly called as homozygous  wheat reference genome with just one , two  or all three (A+B+D) homoeologous copies of the The appearance of homoeologous SNPs at positions 1020 . In theget genes and may –3. To be 95% confident of finding such a null mutation within the EMS-mutagenized population would therefore require the identification of all genic mutations in approximately 1,500 lines. In this pilot scale exon capture experiment the materials and sequencing costs amounted to $200 US for exon capture and $2000 for sequencing. The benefits of scale and improved exon capture technology mean that it is now feasible to create an exon-capture array covering the whole genome at a similar cost. The price for full-genome exon capture and sequencing of 1,500 lines is therefore estimated at $1.1M US which we consider not excessive given the benefits to functional genomics, model-to-crop translation and crop improvement opportunities that would be enabled by such an investment.Analysis of the annotated wheat chromosome 3B suggestsWe have demonstrated the feasibility of using targeted re-sequencing, based on exon capture followed by next generation sequencing, to identify induced mutations in hexaploid bread wheat. A capture array based on coding sequence from one of the homoeologues of the target genes allowed selective enrichment of sequence reads from all three homoeologues, albeit with lower efficiencies for the two off-target copies. Where all three homoeologues are present in the reference sequence, detection of mutations in the homozygous or heterozygous state is straightforward and, if carried out on a genome-wide scale with a large population of EMS mutants, would represent a very valuable resource for functional genomics, hypothesis testing and crop improvement. However, it is clear that the incomplete state of the wheat genome sequence, with many missing genes, creates problems for the alignment of captured reads to the correct genomic target sequence where one or more homoeologues are missing from the reference. Improving the quality, completeness and contiguity of the wheat genome sequence must therefore remain a priority for the worldwide wheat community.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S1 File(FASTA)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.