Publications

2014
Ben-David, E., Shohat, S. & Shifman, S. Allelic expression analysis in the brain suggests a role for heterogeneous insults affecting epigenetic processes in autism spectrum disorders. Hum Mol Genet 23, 4111-24 (2014).Abstract
Monoallelic expression, including genomic imprinting, X-chromosome inactivation and random monoallelic expression of autosomal genes are epigenetic phenomena. Genes that are expressed in a monoallelic way may be more vulnerable to genetic or epigenetic mutations. Thus, comprehensive exploration of monoallelic expression in human brains may shed light on complex brain disorders. Autism-related disorders are known to be associated with imprinted genes on chromosome 15. However, it is not clear whether other imprinted regions or other types of monoallelic expression are associated with autism spectrum disorder (ASD). Here, we performed a genome-wide survey of allele expression imbalance (AEI) in the human brain using single-nucleotide polymorphisms (SNPs), in 18 individuals with ASD and 15 controls. Individuals with ASD had the most extreme number of monoallelic expressed SNPs in both the autosomes and the X chromosome. In two cases that were studied in detail, the monoallelic expression was confined to specific brain region or cell type. Using these data, we were also able to define the allelic expression status of known imprinted genes in the human brain and to identify abnormal imprinting in an individual with ASD. Lastly, we developed an analysis of individual-level expression, focusing on the difference of each individual from the mean. We found that individuals with ASD had more genes that were up- or down-regulated in an individual-specific manner. We also identified pathways perturbed in specific individuals. These results underline the heterogeneity in gene regulation in ASD, at the level of both allelic and total expression.
Shohat, S. & Shifman, S. Bias towards large genes in autism. Nature 512, E1-2 (2014).
Suliman, R., Ben-David, E. & Shifman, S. Chromatin regulators, phenotypic robustness, and autism risk. Front Genet 5, 81 (2014).Abstract
Though extensively characterized clinically, the causes of autism spectrum disorder (ASD) remain a mystery. ASD is known to have a strong genetic basis, but it is genetically very heterogeneous. Recent studies have estimated that de novo disruptive mutations in hundreds of genes may contribute to ASD. However, it is unclear how it is possible for mutations in so many different genes to contribute to ASD. Recent findings suggest that many of the mutations disrupt genes involved in transcription regulation that are expressed prenatally in the developing brain. De novo disruptive mutations are also more frequent in girls with ASD, despite the fact that ASD is more prevalent in boys. In this paper, we hypothesize that loss of robustness may contribute to ASD. Loss of phenotypic robustness may be caused by mutations that disrupt capacitors that operate in the developing brain. This may lead to the release of cryptic genetic variation that contributes to ASD. Reduced robustness is consistent with the observed variability in expressivity and incomplete penetrance. It is also consistent with the hypothesis that the development of the female brain is more robust, and it may explain the higher rate and severity of disruptive de novo mutations in girls with ASD.
Hanin, G., et al. Competing targets of microRNA-608 affect anxiety and hypertension. Hum Mol Genet 23, 4569-80 (2014).Abstract
MicroRNAs (miRNAs) can repress multiple targets, but how a single de-balanced interaction affects others remained unclear. We found that changing a single miRNA-target interaction can simultaneously affect multiple other miRNA-target interactions and modify physiological phenotype. We show that miR-608 targets acetylcholinesterase (AChE) and demonstrate weakened miR-608 interaction with the rs17228616 AChE allele having a single-nucleotide polymorphism (SNP) in the 3'-untranslated region (3'UTR). In cultured cells, this weakened interaction potentiated miR-608-mediated suppression of other targets, including CDC42 and interleukin-6 (IL6). Postmortem human cortices homozygote for the minor rs17228616 allele showed AChE elevation and CDC42/IL6 decreases compared with major allele homozygotes. Additionally, minor allele heterozygote and homozygote subjects showed reduced cortisol and elevated blood pressure, predicting risk of anxiety and hypertension. Parallel suppression of the conserved brain CDC42 activity by intracerebroventricular ML141 injection caused acute anxiety in mice. We demonstrate that SNPs in miRNA-binding regions could cause expanded downstream effects changing important biological pathways.
Barbash, S., Shifman, S. & Soreq, H. Global coevolution of human microRNAs and their target genes. Mol Biol Evol 31, 1237-47 (2014).Abstract
MicroRNAs (miRNAs) have presumably contributed to the emergence of the novel expression patterns, higher brain functions, and skills underlying human evolution. However, it is incompletely understood how new miRNAs have evolved in the human lineage because their initial emergence predictably entailed deleterious consequences due to their powerful multitarget effects. Here, we report genetic variation and conservation parameters for miRNAs and their predicted targets in the genomes of 1,092 humans and 58 additional organisms. We show that miRNAs were evolutionarily more conserved than their predicted binding sites, which were inversely subject to the accumulation of single-nucleotide variations over short evolutionary timescales. Moreover, the predictably "younger" human-specific miRNAs presented lower genetic variation than other miRNAs; their targets displayed higher genetic variation compared with other miRNA targets in diverse human populations; and neuronal miRNAs showed yet lower levels of genetic variation and were found to target more protein-coding genes than nonneuronal miRNAs. Furthermore, enrichment analysis indicated that targets of human-specific miRNAs primarily perform neuronal functions. Specifically, the genomic regions harboring the vertebrate-conserved neuronal miRNA-132 presented considerably higher conservation scores than those of its target genes throughout evolution, whereas both the recently evolved human miRNA-941 and its acquired targets showed relatively low conservation. Our findings demonstrate inversely correlated genetic variation around miRNAs and their targets, consistent with theories of coevolution of these elements and the predicted role attributed to miRNAs in recent human evolution.
Zachor, D.A. & Curatolo, P. Recommendations for early diagnosis and intervention in autism spectrum disorders: an Italian-Israeli consensus conference. Eur J Paediatr Neurol 18, 107-18 (2014).Abstract
On April 2013 experts in the field of autism from Italy and Israel convened in Jerusalem to discuss and finalize clinical recommendations for early diagnosis and intervention in Autism Spectrum Disorders (ASDs). In this paper, we summarize the results of this Italian-Israeli consensus conference. ASDs constitute a class of severe and heterogeneous neurodevelopmental conditions caused by atypical brain development beginning during early prenatal life, reflecting many genetic, neurobiological and environmental influences. The first clinical signs of ASDs begin to be evident in children between 12 and 18 months of age, often after a period of relatively typical postnatal development. Recent longitudinal studies reveal substantial diversity in developmental trajectories through childhood and adolescence. Some intervention approaches have been demonstrated to be effective in improving core symptoms of ASDs, even if the heterogeneity and developmental nature of the disorder make it implausible that only one specific treatment will be best for all children with ASDs. More randomized control trials (RCTs) on early intervention are needed to identify the most effective strategies and provide the most efficient allocation of resources during the critical early intervention time period. Future research should focus on linking biological phenotypes with specific genotypes, thus establishing a foundation for the development of diagnostic screening tools and individualization of treatments.
Ben-David, E., Bester, A.C., Shifman, S. & Kerem, B. Transcriptional dynamics in colorectal carcinogenesis: new insights into the role of c-Myc and miR17 in benign to cancer transformation. Cancer Res 74, 5532-40 (2014).Abstract
Colorectal cancer develops in a sequential, evolutionary process, leading to a heterogenic tumor. Comprehensive molecular studies of colorectal cancer have been previously performed; still, the process of carcinogenesis is not fully understood. We utilized gene expression patterns from 94 samples including normal, adenoma, and adenocarcinoma colon biopsies and performed a coexpression network analysis to determine gene expression trajectories of 8,000 genes across carcinogenesis. We found that the majority of gene expression changes occur in the transition from normal tissue to adenoma. The upregulated genes, known to be involved in cellular proliferation, included c-Myc along with its targets. In a cellular model system, we show that physiologic upregulation of c-Myc can lead to cellular proliferation without DNA replication stress. Our analysis also found that carcinogenesis involves a progressive downregulation of genes that are markers of colonic tissue and propose that this reflects a perturbed differentiation of colon cells during carcinogenesis. The analysis of miRNAs targets pointed toward the involvement of miR17 in the regulation of colon cell differentiation. Finally, we found that copy-number variations (CNV) enriched in colon adenocarcinoma tend to occur in genes whose expression changes already in adenoma, with deletions occurring in genes downregulated and duplications in genes upregulated in adenomas. We suggest that the CNVs are selected to reinforce changes in gene expression, rather than initiate them. Together, these findings shed new light into the molecular processes that underlie the transformation of colon tissue from normal to cancer and add a temporal context that has been hitherto lacking.
2013
Ben-David, E. & Shifman, S. Combined analysis of exome sequencing points toward a major role for transcription regulation during brain development in autism. Mol Psychiatry 18, 1054-6 (2013).
Pandey, V., et al. A new in vivo model of pantothenate kinase-associated neurodegeneration reveals a surprising role for transcriptional regulation in pathogenesis. Front Cell Neurosci 7, 146 (2013).Abstract
Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a neurodegenerative disorder with a poorly understood molecular mechanism. It is caused by mutations in Pantothenate Kinase, the first enzyme in the Coenzyme A (CoA) biosynthetic pathway. Here, we developed a Drosophila model of PKAN (tim-fbl flies) that allows us to continuously monitor the modeled disease in the brain. In tim-fbl flies, downregulation of fumble, the Drosophila PanK homologue in the cells containing a circadian clock results in characteristic features of PKAN such as developmental lethality, hypersensitivity to oxidative stress, and diminished life span. Despite quasi-normal circadian transcriptional rhythms, tim-fbl flies display brain-specific aberrant circadian locomotor rhythms, and a unique transcriptional signature. Comparison with expression data from flies exposed to paraquat demonstrates that, as previously suggested, pathways others than oxidative stress are affected by PANK downregulation. Surprisingly we found a significant decrease in the expression of key components of the photoreceptor recycling pathways, which could lead to retinal degeneration, a hallmark of PKAN. Importantly, these defects are not accompanied by changes in structural components in eye genes suggesting that changes in gene expression in the eye precede and may cause the retinal degeneration. Indeed tim-fbl flies have diminished response to light transitions, and their altered day/night patterns of activity demonstrates defects in light perception. This suggest that retinal lesions are not solely due to oxidative stress and demonstrates a role for the transcriptional response to CoA deficiency underlying the defects observed in dPanK deficient flies. Moreover, in the present study we developed a new fly model that can be applied to other diseases and that allows the assessment of neurodegeneration in the brains of living flies.
2012
Ben-David, E. & Shifman, S. Networks of neuronal genes affected by common and rare variants in autism spectrum disorders. PLoS Genet 8, e1002556 (2012).Abstract
Autism spectrum disorders (ASD) are neurodevelopmental disorders with phenotypic and genetic heterogeneity. Recent studies have reported rare and de novo mutations in ASD, but the allelic architecture of ASD remains unclear. To assess the role of common and rare variations in ASD, we constructed a gene co-expression network based on a widespread survey of gene expression in the human brain. We identified modules associated with specific cell types and processes. By integrating known rare mutations and the results of an ASD genome-wide association study (GWAS), we identified two neuronal modules that are perturbed by both rare and common variations. These modules contain highly connected genes that are involved in synaptic and neuronal plasticity and that are expressed in areas associated with learning and memory and sensory perception. The enrichment of common risk variants was replicated in two additional samples which include both simplex and multiplex families. An analysis of the combined contribution of common variants in the neuronal modules revealed a polygenic component to the risk of ASD. The results of this study point toward contribution of minor and major perturbations in the two sub-networks of neuronal genes to ASD risk.
Brunschwig, H., et al. Fine-scale maps of recombination rates and hotspots in the mouse genome. Genetics 191, 757-64 (2012).Abstract
Recombination events are not uniformly distributed and often cluster in narrow regions known as recombination hotspots. Several studies using different approaches have dramatically advanced our understanding of recombination hotspot regulation. Population genetic data have been used to map and quantify hotspots in the human genome. Genetic variation in recombination rates and hotspots usage have been explored in human pedigrees, mouse intercrosses, and by sperm typing. These studies pointed to the central role of the PRDM9 gene in hotspot modulation. In this study, we used single nucleotide polymorphisms (SNPs) from whole-genome resequencing and genotyping studies of mouse inbred strains to estimate recombination rates across the mouse genome and identified 47,068 historical hotspots--an average of over 2477 per chromosome. We show by simulation that inbred mouse strains can be used to identify positions of historical hotspots. Recombination hotspots were found to be enriched for the predicted binding sequences for different alleles of the PRDM9 protein. Recombination rates were on average lower near transcription start sites (TSS). Comparing the inferred historical recombination hotspots with the recent genome-wide mapping of double-strand breaks (DSBs) in mouse sperm revealed a significant overlap, especially toward the telomeres. Our results suggest that inbred strains can be used to characterize and study the dynamics of historical recombination hotspots. They also strengthen previous findings on mouse recombination hotspots, and specifically the impact of sequence variants in Prdm9.
Huang, G.-J., et al. Neurogenomic evidence for a shared mechanism of the antidepressant effects of exercise and chronic fluoxetine in mice. PLoS One 7, e35901 (2012).Abstract
Several different interventions improve depressed mood, including medication and environmental factors such as regular physical exercise. The molecular pathways underlying these effects are still not fully understood. In this study, we sought to identify shared mechanisms underlying antidepressant interventions. We studied three groups of mice: mice treated with a widely used antidepressant drug--fluoxetine, mice engaged in voluntary exercise, and mice living in an enriched environment. The hippocampi of treated mice were investigated at the molecular and cellular levels. Mice treated with fluoxetine and mice who exercised daily showed, not only similar antidepressant behavior, but also similar changes in gene expression and hippocampal neurons. These changes were not observed in mice with environmental enrichment. An increase in neurogenesis and dendritic spine density was observed following four weeks of fluoxetine treatment and voluntary exercise. A weighted gene co-expression network analysis revealed four different modules of co-expressed genes that were correlated with the antidepressant effect. This network analysis enabled us to identify genes involved in the molecular pathways underlying the effects of fluoxetine and exercise. The existence of both neuronal and gene expression changes common to antidepressant drug and exercise suggests a shared mechanism underlying their effect. Further studies of these findings may be used to uncover the molecular mechanisms of depression, and to identify new avenues of therapy.
2011
Ovadia, G. & Shifman, S. The genetic variation of RELN expression in schizophrenia and bipolar disorder. PLoS One 6, e19955 (2011).Abstract
Reelin plays an important role in the development and function of the brain and has been linked to different neuropsychiatric diseases. To further clarify the connection between reelin and psychiatric disorders, we studied the factors that influence the expression of reelin gene (RELN) and its different isoforms. We examined the total expression of RELN, allelic expression, and two alternative RELN isoforms in postmortem brain samples from patients with schizophrenia and bipolar disorder, as well as unaffected controls. We did not find a significant reduction in the total expression of RELN in schizophrenia or bipolar disorder. However, we did find a significant reduction of the proportion of the short RELN isoform, missing the C-terminal region in bipolar disorder, and imbalance in the allelic expression of RELN in schizophrenia. In addition, we tested the association between variation in RELN expression and rs7341475, an intronic SNP that was found to be associated with schizophrenia in women. We did not find an association between rs7341474 and the total expression of RELN either in women or in the entire sample. However, we observed a nominally significant effect of genotype-by-sex interaction on the variation in microexon skipping. Women with the risk genotype of rs7341475 (GG) had a higher proportion of microexon skipping, which is the isoform predominant in tissues outside the brain, while men had the opposite trend. Finally, we tested 83 SNPs in the gene region for association with expression variation of RELN, but none were significant. Our study further supports the connection between RELN dysfunction and psychiatric disorders, and provides a possible functional role for a schizophrenia associated SNP. Nevertheless, the positive associations observed in this study needs further replication as it may have implications for understanding the biological causes of schizophrenia and bipolar disorder.
Ben-David, E., et al. Identification of a functional rare variant in autism using genome-wide screen for monoallelic expression. Hum Mol Genet 20, 3632-41 (2011).Abstract
Recent work has led to the identification of several susceptibility genes for autism spectrum disorder (ASD) and an increased appreciation of the importance of rare and de novo mutations. Some of the mutations may be very hard to detect using current strategies, especially if they are located in regulatory regions. We present a new approach to identify functional mutations that exploit the fact that many rare mutations disrupt the expression of genes from a single parental chromosome. The method incorporates measurement of the relative expression of the two copies of a gene across the genome using single nucleotide polymorphism arrays. Allelic expression has been successfully used to study common regulatory polymorphisms; however, it has not been implemented as a screening tool for rare mutation. We tested the potential of this approach by screening for monoallelic expression in lymphoblastoid cell lines derived from a small ASD cohort. After filtering regions shared across multiple samples, we identified genes showing monoallelic expression in specific ASD samples. Validation by quantitative sequencing demonstrated that the genes (or only part of them) are monoallelic expressed. The genes included both previously suspected risk factors for ASD and novel candidates. In one gene, named autism susceptibility candidate 2 (AUTS2), we identified a rare duplication that is likely to be the cause of monoallelic expression. Our results demonstrate the ability to identify rare regulatory mutations using genome-wide allelic expression screens, capabilities that could be expanded to other diseases, especially those with suspected involvement of rare dominantly acting mutations.
2010
Ben-David, E. & Shifman, S. Further investigation of the association between rs7341475 and rs17746501 and schizophrenia. Am J Med Genet B Neuropsychiatr Genet 153B, 1244-7 (2010).Abstract
Genome-wide association studies (GWAS) with small sample size have had limited statistical power in identifying schizophrenia susceptibility genes. This is exemplified by the fact that one of the most convincing associations was detected only after meta-analyses of three different GWAS. Here we used meta-analysis to study the association of two single-nucleotide polymorphisms (SNPs) (rs7341475 and rs17746501) previously indicated to be associated with schizophrenia by a GWAS of Ashkenazi Jews (AJ). In the initial report, rs7341475 was associated only in women, while rs17746501 was associated in both men and women. We collected genotyping results of samples published in four GWAS for the two SNPs, additional to results from AJ. We used the Mantel-Haenszel method to combine the data of the different samples. For both SNPs, the results of the meta-analysis of all samples, including the initial report, did not reach a genome-wide significance level. However, the association between rs7341475 and schizophrenia in women, after excluding the data from AJ, was significant (P = 9.0 x 10(-3)), with a calculated odds ratio (OR) of 1.11, much smaller than the original result. Association between rs17746501 and schizophrenia was significant in four of the new samples, showing evidence for heterogeneity and very small effect when tested across all samples (P = 0.016, OR = 1.06). These findings suggest that the two SNPs might have a small effect on schizophrenia risk and suggest that meta-analyses of very large samples are needed to adequately study the contribution of common variants to schizophrenia susceptibility.
Narwani, K., et al. Human embryonic stem cells from aneuploid blastocysts identified by pre-implantation genetic screening. In Vitro Cell Dev Biol Anim 46, 309-16 (2010).Abstract
Human embryonic stem cells are derived from the inner cell mass of pre-implantation embryos. The cells have unlimited proliferation potential and capacity to differentiate into the cells of the three germ layers. Human embryonic stem cells are used to study human embryogenesis and disease modeling and may in the future serve as cells for cell therapy and drug screening. Human embryonic stem cells are usually isolated from surplus normal frozen embryos and were suggested to be isolated from diseased embryos detected by pre-implantation genetic diagnosis. Here we report the isolation of 12 human embryonic stem cell lines and their thorough characterization. The lines were derived from embryos detected to have aneuploidy by pre-implantation genetic screening. Karyotype analysis of these cell lines showed that they are euploid, having 46 chromosomes. Our interpretation is that the euploid cells originated from mosaic embryos, and in vitro selection favored the euploid cells. The undifferentiated cells exhibited long-term proliferation and expressed markers typical for embryonic stem cells such as OCT4, NANOG, and TRA-1-60. The cells manifested pluripotent differentiation both in vivo and in vitro. To further characterize the different lines, we have analyzed their ethnic origin and the family relatedness among them. The above results led us to conclude that the aneuploid mosaic embryos that are destined to be discarded can serve as source for normal euploid human embryonic stem cell lines. These lines represent various ethnic groups; more lines are needed to represent all populations.
2009
O'Donovan, M.C., et al. Analysis of 10 independent samples provides evidence for association between schizophrenia and a SNP flanking fibroblast growth factor receptor 2. Mol Psychiatry 14, 30-6 (2009).Abstract
We and others have previously reported linkage to schizophrenia on chromosome 10q25-q26 but, to date, a susceptibility gene in the region has not been identified. We examined data from 3606 single-nucleotide polymorphisms (SNPs) mapping to 10q25-q26 that had been typed in a genome-wide association study (GWAS) of schizophrenia (479 UK cases/2937 controls). SNPs with P<0.01 (n=40) were genotyped in an additional 163 UK cases and those markers that remained nominally significant at P<0.01 (n=22) were genotyped in replication samples from Ireland, Germany and Bulgaria consisting of a total of 1664 cases with schizophrenia and 3541 controls. Only one SNP, rs17101921, was nominally significant after meta-analyses across the replication samples and this was genotyped in an additional six samples from the United States/Australia, Germany, China, Japan, Israel and Sweden (n=5142 cases/6561 controls). Across all replication samples, the allele at rs17101921 that was associated in the GWAS showed evidence for association independent of the original data (OR 1.17 (95% CI 1.06-1.29), P=0.0009). The SNP maps 85 kb from the nearest gene encoding fibroblast growth factor receptor 2 (FGFR2) making this a potential susceptibility gene for schizophrenia.
Huang, G.-J., et al. High resolution mapping of expression QTLs in heterogeneous stock mice in multiple tissues. Genome Res 19, 1133-40 (2009).Abstract
A proportion of the genetic variants underlying complex phenotypes do so through their effects on gene expression, so an important challenge in complex trait analysis is to discover the genetic basis for the variation in transcript abundance. So far, the potential of mapping both quantitative trait loci (QTLs) and expression quantitative trait loci (eQTLs) in rodents has been limited by the low mapping resolution inherent in crosses between inbred strains. We provide a megabase resolution map of thousands of eQTLs in hippocampus, lung, and liver samples from heterogeneous stock (HS) mice in which 843 QTLs have also been mapped at megabase resolution. We exploit dense mouse SNP data to show that artifacts due to allele-specific hybridization occur in approximately 30% of the cis-acting eQTLs and, by comparison with exon expression data, we show that alternative splicing of the 3' end of the genes accounts for <1% of cis-acting eQTLs. Approximately one third of cis-acting eQTLs and one half of trans-acting eQTLs are tissue specific. We have created an important systems biology resource for the genetic analysis of complex traits in a key model organism.
2008
Shifman, S., et al. A whole genome association study of neuroticism using DNA pooling. Mol Psychiatry 13, 302-12 (2008).Abstract
We describe a multistage approach to identify single nucleotide polymorphisms (SNPs) associated with neuroticism, a personality trait that shares genetic determinants with major depression and anxiety disorders. Whole genome association with 452 574 SNPs was performed on DNA pools from approximately 2000 individuals selected on extremes of neuroticism scores from a cohort of 88 142 people from southwest England. The most significant SNPs were then genotyped on independent samples to replicate findings. We were able to replicate association of one SNP within the PDE4D gene in a second sample collected by our laboratory and in a family-based test in an independent sample; however, the SNP was not significantly associated with neuroticism in two other independent samples. We also observed an enrichment of low P-values in known regions of copy number variations. Simulation indicates that our study had approximately 80% power to identify neuroticism loci in the genome with odds ratio (OR)>2, and approximately 50% power to identify small effects (OR=1.5). Since we failed to find any loci accounting for more than 1% of the variance, the heritability of neuroticism probably arises from many loci each explaining much less than 1%. Our findings argue the need for much larger samples than anticipated in genetic association studies and that the biological basis of emotional disorders is extremely complex.
Shifman, S., et al. Genome-wide association identifies a common variant in the reelin gene that increases the risk of schizophrenia only in women. PLoS Genet 4, e28 (2008).Abstract
Sex differences in schizophrenia are well known, but their genetic basis has not been identified. We performed a genome-wide association scan for schizophrenia in an Ashkenazi Jewish population using DNA pooling. We found a female-specific association with rs7341475, a SNP in the fourth intron of the reelin (RELN) gene (p = 2.9 x 10(-5) in women), with a significant gene-sex effect (p = 1.8 x 10(-4)). We studied rs7341475 in four additional populations, totaling 2,274 cases and 4,401 controls. A significant effect was observed only in women, replicating the initial result (p = 2.1 x 10(-3) in women; p = 4.2 x 10(-3) for gene-sex interaction). Based on all populations the estimated relative risk of women carrying the common genotype is 1.58 (p = 8.8 x 10(-7); p = 1.6 x 10(-5) for gene-sex interaction). The female-specific association between RELN and schizophrenia is one of the few examples of a replicated sex-specific genetic association in any disease.

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