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• Smoking and the Vaginal Microbiome

  The focus of this study was to better understand the effects of cigarette smoking on the vaginal microbiome. There were two phases of the study, cross-sectional and longitudinal, conducted at the Center for Health Behavior Research at the University of Maryland School of Public Health. In the cross-sectional phase, 20 smokers and 20 non-smokers collected mid-vaginal swabs, measured their vaginal pH, prepared a vaginal smear on a slide for Nugent Gram stain analysis, and completed questionnaires about demographics, tobacco use, and reproductive and sexual health history. Smoking status was confirmed through self-report, carbon monoxide exhalation and saliva cotinine measures. Secretions from the mid-vaginal swabs were tested for presence/absence of HPV strains and GC-MS was used to quantify the levels of over 600 metabolites. In the longitudinal phase, 7 participants who were current smokers and motivated to quit smoking were recruited and followed for up to 12 weeks. On a daily basis, participants collected mid-vaginal swabs, measured their vaginal pH, and prepared a mid-vaginal smear on a slide for Nugent Gram stain analysis, and completed daily diaries on tobacco use and reproductive health. Carbon monoxide exhalation and saliva cotinine measures were collected at weekly clinical visits. In addition, participants had weekly behavioral counseling sessions about smoking cessation and used Nicoderm CQ patches to aid in quitting smoking. The self-collected vaginal swabs were used for DNA extractions,16s rRNA sequencing and measurement of metabolites in vaginal fluid.

  Study phs001386

• Tumor Mutation Burden, Expressed Neoantigen and Immune Microenvironment in Diffuse Gliomas

  A consistent correlation between tumor mutation burden (TMB) and tumor immune microenvironment has not been observed in gliomas as in other cancers. Driver germline and somatic mutations, TMB, neoantigen, and immune cell signatures were analyzed using whole exome sequencing (WES) and transcriptome sequencing of tumor and WES of matched germline DNA in a cohort of 66 glioma samples (44 IDH-mutant and 22 IDH-wildtype). Fourteen samples revealed a hypermutator phenotype (HMP). Eight pathogenic (P) or likely pathogenic (LP) germline variants were detected in 9 (19%) patients. Six of these 8 genes were DNA damage repair genes. P/LP germline variants were found in 22% of IDH-mutant gliomas and 12.5%of IDH-wildtype gliomas (p= 0.7). TMB was correlated with expressed neoantigen but showed an inverse correlation with immune score (R =−0.46, p= 0.03) in IDH-wildtype tumors and no correlation in IDH-mutant tumors. Antigen Processing and Presentation (APP) score correlates with immune score. The APP is higher in non-hypermutator phenotype (NHMP) samples versus HMP samples in the IDH-wildtype gliomas, but higher in HMP samples versus NHMP samples in the IDH-mutant gliomas. Conclusion: TMB was inversely correlated with the immune score in the IDH-wildtype gliomas, and showed no correlation in the IDH-mutant tumors. The APP was correlated with the immune score, and may be further investigated as a biomarker for response to immunotherapy in gliomas. Studies of germline variants in a large glioma cohort are warranted.

  Study phs002653

• Region-Specific Neural Stem Cell Lineages Revealed by Single-Cell RNA-Sequences from Human Embryonic Stem Cells

  During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially be generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development.

  Study phs001205

• Cell Type-Specific and Disease-Associated eQTL in the Human Lung

  This study was conducted to investigate the genetic control of gene expression in a cell-type and context-specific manner in the human lung. We performed single-cell RNA-sequencing (scRNA-seq) of lung tissue from 66 Interstitial Lung Disease (ILD) cases and 48 unaffected donors. Employing a pseudo-bulk approach, we mapped expression quantitative trait loci (eQTL) across 38 cell types. The scRNA-seq data presented here include previously published (PMID 34262047) and unpublished samples. ILD tissue samples were obtained from lungs removed at the time of lung transplantation at either Vanderbilt University Medical Center (VUMC) or the Norton Thoracic Institute (NTI). Control tissue samples were obtained from lungs declined for organ donation either at the Donor Network of Arizona (DNA) or VUMC. The self-reported ethnicities of donors were 66.7% European, 9.6% African American, 17.5% not available (N/A), and 6.1% other. The smoking history includes 46.5% ever smoker, 29.8% never smoker, 23.7% N/A. For genotyping, flash-frozen tissue in DNA/RNA shield was homogenized via a bullet blender. Genomic DNA was extracted using Zymo Quick DNA/RNA microprep plus kit. Library preparation and low-pass Whole Genome Sequencing (WGS) were carried out at TGen or by Gencove Inc. At TGen, libraries were prepared using PCR-free Watchmaker kits (Watchmaker Genomics) with 200 ng input. Genomes were sequenced on NovaSeq at low coverage (typically 0.4–1x). The resulting sequence data were processed and imputed using Gencove's imputation platform.

  Study phs003521

• Uncovering Inversion Formation in the Human Genome and its Impact to Disease

  The relevance of inversions for disease causation, speciation and adaptation is broadly recognized, although the prevalence of inversions is unknown. In humans, de novo inversions are associated with congenital anomalies in approximately 9.6% of patients. Yet, despite the biological relevance of inversions, their molecular features, formation mechanism, impact to the genomic structure of carriers, as well as their contribution to clinical phenotypes, have not been further explored. Inversions are typically classified as balanced reciprocal events generated by ectopic recombination, although recent studies reveal a distinct picture whereby inversions originate from mechanisms that concomitantly generate copy number variants (CNVs). Surprisingly, those complex inversions underlie as much as 30% of neurodevelopmental-associated CNVs. The hypothesis of this project are: inversions are often generated de novo by mechanisms other than ectopic recombination a relevant fraction of inversions are associated with complex genomic rearrangements (CGRs), often overlooked in sporadic diseases, and inversions are a "hidden" type of structural variation for which contribution to a clinical phenotypes has been under assessed due to the lack of appropriate detection tools. A combined strategy using multiple genomic tools will be applied to characterize inversions and associated genomic alterations in individuals with neurodevelopmental disorders and controls: whole genome sequencing (WGS) with short-and long-reads; genome mapping; classical cytogenetics; and array CGH. This project will establish common ground to bridge studies of rare and common diseases, human evolution, and population genetics.

  Study phs002999

• Risk Factors for Asymptomatic Diverticulosis

  Data for this analysis came from a prospective study of colonic diverticulosis at the University of North Carolina Hospital in Chapel Hill, North Carolina (NIH R01DK094738). This is a case-control genetic and transcriptomic study to identify the genetic and cellular determinants underlying colonic diverticulosis and the relationship with other gastrointestinal disorders. The study inclusion and exclusion are described below. DNA and RNA sequencing were performed on colonic tissue from 404 patients with (N=172) and without (N=232) diverticulosis. We assessed variation in the transcriptome associated with diverticulosis and integrated this variation with human intestine single-cell RNA-seq data. We integrated our expression quantitative trait loci with GWAS using Mendelian Randomization. A polygenic risk score analysis examined associations between severity of diverticulosis and other gastrointestinal disorders. We identified 38 genes with differential expression and 17 genes with varied transcript usage linked to diverticulosis. Our results suggest tissue remodeling as a primary mechanism for diverticula formation. The presence of diverticulosis was linked to stromal and epithelial cells in the colon including endothelial cells, myofibroblasts, fibroblasts, goblet, tuft, enterocytes, neurons, and glia. MR highlighted five genes including CCN3, CRISPLD2, ENTPD7, PHGR1, and TNFSF13. In diverticulosis cases, ENTPD7 upregulation was confirmed. A greater burden of diverticula was positively correlated with genetic predisposition to diverticulitis. The data related to the study including total RNA-Seq, SNP array and imputed genotypes will be available through dbGaP.

  Study phs003556

• Alpha1-Antitrypsin Deficiency Registry (AADR-BioLINCC)

  Data Access NOTE: Please refer to the “Authorized Access” section below for information about how access to the data from this accession differs from many other dbGaP accessions. Objectives: To characterize the clinical and laboratory course of patients with severe alpha 1-antitrypsin deficiency whether or not the patient is undergoing long-term augmentation therapy.Background: A hereditary disorder, patients with low serum levels of alpha-1-antitrypsin are at an increased risk for the early onset of emphysema. The only approved treatment for alpha-1-antitrypsin deficiency is augmentation therapy using a purified preparation of human alpha-1-antitryspin. Sample sizes for a randomized controlled clinical trial of augmentation therapy were determined to be infeasible; therefore, a multi-center registry was initiated in 1988 to explore the natural history of the disease and the relative efficacy of augmentation therapy in patients with a severe deficiency of alpha-1-antitrypsin.Participants: Eligible participants included individuals 18 years of age or greater for whom the Central Laboratory confirmed that the serum alpha 1-antitrypsin level is Conclusions: Participants receiving augmentation therapy had decreased mortality risk during follow-up. FEV1 decline among all participants did not differ by augmentation therapy; however, among participants with FEV1 35-49% predicted, FEV1 decline was significantly slower for participants on augmentation therapy than for those not receiving therapy. (Am J Respir Crit Care Med, 1998; 158:49-59)

  Study phs004187

• Whole exome sequencing data for paired non-serous endometrial and ovarian carcinomas from 27 patients with concurrent tumours.

  Introduction: Concurrent non-serous endometrial and ovarian tumours are often managed clinically as two separate primary tumours, but almost all exhibit evidence of a genomic relationship. Methodology: This study investigates the extent of relatedness using whole exome sequencing, which was performed on paired non-serous endometrial and ovarian carcinomas from 27 patients with concurrent tumours in a cohort with detailed clinicopathological annotation. Four whole exome sequencing-derived parameters (mutation, mutational burden, mutational signatures and mutant allele tumour heterogeneity scores) were used to develop a novel unsupervised model for the assessment of genomic similarity to infer genomic relatedness of paired tumours. Results: This novel model demonstrated genomic relatedness across all four parameters in all tumour pairs. Mutations in PTEN, ARID1A, CTNNB1, KMT2D and PIK3CA occurred most frequently and 24 of 27 (89 %) tumour pairs shared identical mutations in at least one of these genes, with all pairs sharing mutations in a number of other genes. Ovarian endometriosis, CTNNB1 exon 3 mutation, and progression and death from disease were present across the similarity ranking. Mismatch repair deficiency was associated with less genomically similar pairs. Discussion: Although there was diversity across the cohort, the presence of genomic similarity in all paired tumours supports the hypothesis that concurrent non-serous endometrial and ovarian carcinomas are genomically related and may have arisen from a common origin. Keywords: Co-existing; Concurrent; Endometrial carcinoma; Genomics; Mutation; Ovarian carcinoma; Paired tumours; Tumour mutational burden.

  Study EGAS00001008259

• Evolution of the African pygmy phenotype

  The evolutionary history of the human ‘pygmy’ phenotype (small body size), a characteristic of African and Southeast Asian rainforest hunter-gatherers, is largely unknown. Here we use a genome-wide admixture mapping analysis to identify 16 genomic regions that are significantly associated with the pygmy phenotype in the Batwa, a rainforest hunter-gatherer population from Uganda (East Central Africa). The identified genomic regions have multiple attributes that provide supporting evidence of genuine association with the pygmy phenotype, including enrichments for SNPs previously associated with stature variation in Europeans and for genes with growth hormone receptor and regulation functions. To test adaptive evolutionary hypotheses we computed the haplotype-based iHS statistic and the level of population differentiation (FST) between the Batwa and their agricultural neighbors, the Bakiga, for each SNP. Both |iHS| and FST values were significantly higher for SNPs within the Batwa pygmy phenotype-associated regions than the remainder of the genome, a signature of polygenic adaptation. In contrast, when we expanded our analysis to include Baka rainforest hunter-gatherers from Cameroon and Gabon (West Central Africa) and Nzebi and Nzime neighboring agriculturalists, we did not observe elevated |iHS| or FST values in these genomic regions. Together, these results suggest adaptive and at least partially convergent origins of the pygmy phenotype even within Africa, supporting the hypothesis that small body size confers a selective advantage for tropical rainforest hunter-gatherers but raising questions about the antiquity of this behavior.

  Study EGAS00001000908

• Platelet RNAseq data for SLFN14 K219N patients

  Pathogenic missense variants in SLFN14, which encodes an RNA endoribonuclease protein that regulates rRNA degradation, are known to cause inherited thrombocytopenia with impaired platelet aggregation and ATP secretion. Despite rather mild laboratory defects, these patients display an obvious bleeding phenotype. The function of SLFN14 in megakaryocyte (MK) and platelet biology is unknown. This study aims to characterize the platelet transcriptome in patients with a SLFN14 K219N variant and model the disease in the immortalized megakaryocyte cell line imMKCL. Total platelet RNA was sequenced for two patients and 19 healthy controls. Differential gene expression analysis yielded a total of 2999 and 2888 significantly (|log2FC|>1, FDR<0.05) up- and downregulated genes, respectively. Remarkably, these downregulated genes were not enriched for any biological pathway while upregulated genes were enriched for pathways involved in (mitochondrial) translation and transcription with a significant upregulation of 134 ribosomal protein genes (RPG). Heterozygous and homozygous SLFN14 K219N imMKCL showed a defect in MK differentiation that aggravated during cell passaging of undifferentiated cells and in proplatelet formation. SLFN14 defective platelets and MK showed signs of rRNA degradation while this was absent in undifferentiated imMKCL cells. Upregulation of RPG through increased mTOR signaling in SLFN14 K219N MK seems to be a compensatory response for rRNA degradation. Indeed, mTOR inhibition with rapamycin resulted in further enhanced rRNA degradation in SLFN14 K219N MK. Taken together, our study indicates dysregulation of mTOR-regulated ribosomal biogenesis as the disease mechanism for SLFN14-related thrombocytopenia.

  Study EGAS00001006339

• RB1 Gene Inactivation by Chromothripsis in Human Retinoblastoma

  Retinoblastoma is a rare childhood cancer of the developing retina. Most retinoblastomas initiate with biallelic inactivation of the RB1 gene through diverse mechanisms including point mutations, nucleotide insertions, deletions, loss of heterozygosity and promoter hypermethylation. Recently, a novel mechanism of retinoblastoma initiation was proposed. Gallie and colleagues discovered that a small proportion of retinoblastomas lack RB1 mutations and had MYCN amplification [1]. In this study, we identified recurrent chromosomal, regional and focal genomic lesions in 94 primary retinoblastomas with their matched normal DNA using SNP 6.0 chips. We also analyzed the RB1 gene mutations and compared the mechanism of RB1 inactivation to the recurrent copy number variations in the retinoblastoma genome. In addition to the previously described focal amplification of MYCN and deletions in RB1 and BCOR, we also identified recurrent focal amplification of OTX2, a transcription factor required for retinal photoreceptor development. We identified 10 retinoblastomas in our cohort that lacked RB1 point mutations or indels. We performed whole genome sequencing on those 10 tumors and their corresponding germline DNA. In one of the tumors, the RB1 gene was unaltered, the MYCN gene was amplified and RB1 protein was expressed in the nuclei of the tumor cells. In addition, several tumors had complex patterns of structural variations and we identified 3 tumors with chromothripsis at the RB1 locus. This is the first report of chromothripsis as a mechanism for RB1 gene inactivation in cancer.

  Study EGAS00001000598

• Bacterial SNPs in the human gut microbiome associate with host BMI

  Genome-wide association studies (GWAS) have revolutionized the field of genetics, providing numerous associations between human SNPs and health traits. Likewise, metagenome-wide association studies (MWAS) between bacterial SNPs and human traits can suggest mechanistic links between the microbiota and its host. However, very few such studies have been done to date, in part due to the large number of samples required to address the variable coverage of bacteria across samples and the fact that most bacteria are present in only a subset of the population. Here, we devised an MWAS framework to detect SNPs and associate them with host phenotypes systematically. We recruited and obtained gut metagenomic samples from a cohort of 7,190 healthy individuals and discovered 1,358 statistically significant associations between a bacterial SNP and host body mass index (BMI). To address the possible effects of population structure and linkage disequilibrium, we applied a clumping procedure, and found 40 independent associations between SNPs and host BMI. We uncovered BMI-associated SNPs in the genomes of 27 bacterial species, even though the relative abundance of 12 of these species was not associated with the phenotype. We separated genome-wide sub-species variations from local associations in individual SNPs, and demonstrated how this framework can highlight specific bacterial functions of interest. Our results demonstrate the importance of considering nucleotide-level diversity in microbiome studies and pave the way toward a better understanding of interpersonal gut microbiome differences and their health implications.

  Study EGAS00001007204

• Molecular Attributes Underlying Central Nervous System and Systemic Relapse in Diffuse Large B-cell Lymphoma

  Central nervous system (CNS) relapse in diffuse large B-cell lymphoma remains a significant conundrum. While clinical risk scores and biomarkers allow the identification of patients with higher risk of CNS relapse, their predictive ability is low. Herein, we report findings from a comprehensive study examining gene expression changes, mutations and clonal evolution. Firstly, considering diagnostic samples from patients with CNS relapse, systemic relapse or no relapse, we found a striking enrichment of gene expression changes reflecting protein biosynthesis in patients who experienced CNS or systemic relapse. Furthermore, diagnostic specimens of relapsing cases were characterized by low immune infiltrates. The most discriminative gene feature in our analysis was SLAMF1, high expression of which was associated with favourable outcomes. Next, we assessed whether specific gene mutations were associated with subsequent CNS or systemic relapse. Activated B-cell-like genes such as PIM1, CD79B and PRDM1 were more frequently mutated in cases with CNS relapse, whereas TP53, BCL2 and FOXO1 mutations were associated with systemic relapse. Lastly, we performed tumor evolution analysis in five patients, identifying significant clonal divergence that accompanied CNS relapse. In summary, we identified both common biology underlying both CNS and systemic relapse, independent of cell-of-origin (COO), and specific COO-enriched gene mutations that conferred a higher risk of either CNS or systemic relapse. Moreover, we identified a pattern of clonal evolution that can explain difficulties in both predicting and treating CNS relapse.

  Study EGAS00001004057

• UK10K_RARE_HYPERCHOL

  In the UK10K project we propose a series of complementary genetic approaches to find new low frequency/rare variants contributing to disease phenotypes. These will be based on obtaining the genome wide sequence of 4000 samples from the TwinsUK and ALSPAC cohorts (at 6x sequence coverage), and the exome sequence (protein coding regions and related conserved sequence) of 6000 samples selected for extreme phenotypes. Our studies will focus primarily on cardiovascular-related quantitative traits, obesity and related metabolic traits, neurodevelopmental disorders and a limited number of extreme clinical phenotypes that will provide proof-of-concept for future familial trait sequencing. We will analyse directly quantitative traits in the cohorts and the selected traits in the extreme samples, and also use imputation down to 0.1% allele frequency to extend the analyses to further sample sets with genome wide genotype data. In each case we will investigate indels and larger structural variants as well as SNPs, and use statistical methods that combine rare variants in a locus or pathway as well as single-variant approaches.Familial Hypercholesterolemia is a condition where the affected person has a consistently high level of LDL which can lead to early clogging of the coronary arteries. All patients selected for this study will have been found not to carry the common APOB and PCSK9 mutations, and to have no detectable LDLR mutations by testing for 18 common mutations. For further information with regard to this cohort please contact Steve Humphries(steve.humphries@ucl.ac.uk).

  Study EGAS00001000129

• Preclinical Pediatric Molecular Analysis for Therapy Choice (MATCH)

  Purpose: Pediatric solid tumors arise from diverse tissues during development and exhibit a wide range of molecular, cellular and genetic features. This diversity, combined with the low incidence of pediatric cancer makes it increasingly difficult to personalize therapy for individual patients based on the unique features of their tumors. Therefore, well-credentialed preclinical models that capture the diversity and heterogeneity of pediatric solid tumors are essential for identifying molecular targeted therapeutics for precision medicine. Experimental Design: Here, we report 281 orthotopic patient derived xenografts (O-PDXs) from 224 patients representing 24 different types of pediatric solid tumors. We have performed genomic characterization of the O-PDXs and compared them to their corresponding patient tumors. To demonstrate the feasibility and utility of using such a diverse collection of O-PDXs in preclinical studies, we performed a preclinical pediatric precision medicine trial based on the NCI-COG Pediatric MATCH trial enrollment criteria. We also tested molecular targeted therapy for a novel oncogenic fusion recently reported in pediatric melanoma and precision drug delivery using nano-liposomal irinotecan. Results: In the preclinical match study, we identified one specific single agent response to LY3023414 in an osteosarcoma model with a TSC2 variant of unknown significance. Combinations of nano-liposomal irinotecan were effective in treating both Ewing sarcoma and rhabdomyosarcoma xenografts. Conclusion: Our studies demonstrate the value of large, well-credentialed preclinical models for future precision medicine in pediatric oncology using single agents, drug combinations and novel drug formulations.

  Study EGAS00001008011

• Genetic and epigenetic characterization of adenoid cystic carcinoma

  Translocation events are frequent in cancer and may create chimeric fusions or regulatory rearrangements that drive oncogene overexpression. Although regulatory rearrangements are increasingly recognized in hematopoietic and even solid tumors, the underlying mechanisms remain obscure. Here we identify super-enhancer translocations that drive overexpression of the oncogenic transcription factor MYB as a unifying theme in adenoid cystic carcinoma (ACC). Whole genome sequencing data and chromatin state maps for 13 primary tumors and xenografts reveal distinct chromosomal rearrangements that juxtapose super-enhancer clusters to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers physically interact with the MYB promoter. Remarkably, the MYB product itself binds to the translocated enhancers, thus creating a positive feedback loop that sustains its own expression. MYB also binds a large number of active enhancers that drive opposing regulatory programs in alternate cell lineages in ACC. MYB cooperates with the transcription factor TP63 in the myoepithelial component of the tumors, but promotes a Notch program in the luminal epithelial component. Bromodomain inhibitors, which disrupt enhancer function, slow tumor growth in ACC primagraft models in vivo, but are ineffective against high grade tumors that harbor activating mutations in the Notch pathway. Thus, our study identifies super-enhancer translocations as a unifying feature of ACC, and provides insight into the mechanism by which sustained MYB overexpression drives alternate cell fates in this disease.Processed ChIP-seq data is available on GEO under accession number GSE76465.

  Study EGAS00001001457

• Whole Tumor spatial heterogeneity in metastatic melanoma

  Heterogeneous inter-tumoral responses, sustained periods of apparent clinical benefit despite lack of objective response to various therapies, and even spontaneous remission are well known within a subpopulation of advanced melanoma patients. The molecular and cellular dynamics facilitating long-term survival remain poorly defined, particularly in the current era ofexposure to multiple potentially active therapies. We studied an exceptional case of long-term survival in a patient with non-responding metastatic melanoma in order to characterize the clonal and microenvironmental factors.We performed immunogenomic analysis of 3 metachronous tumors, and of 67 intratumor sub-regions of one metastatic lesion, usingwhole exome sequencing, RNA-sequencing, immunohistochemistry, and T cell receptor sequencing. Longitudinal analysesidentified mutations in severalknownresistance genes affecting distinctmetastases. Intra-tumoral immune signatures revealed spatially-distinct pockets of immune activation and suppression throughout the metastasis. A specific T cell VBeta CDR3 rearrangement was identified as both dominant and recurrent not only across multiple spatial points within a single tumor mass, but also across metachronous tumors spanning the patient’s disease course. Immunophenotyping of this T cell population with single-cell sequencingsuggestedrepeatedT-cell priming events leading to the persistence of both activated and exhausted T cells bearing this same TCR-Beta at any given time.Our findings highlight genomic and immune heterogeneityin metastatic disease, extending to the intra-tumoral leveland provide a rationale for integrative spatiotemporal analysis of metastatic lesions in the context of immune checkpoint blockade.

  Study EGAS00001003292

• Atypical 3q26/MECOM rearrangements genocopy inv(3)/t(3;3) in acute myeloid leukemia

  Acute myeloid leukemia (AML) with inv(3)/t(3;3)(q21q26) is a distinct WHO recognized entity, characterized by its aggressive course and poor prognosis. In this subtype of AML, the translocation of a GATA2 enhancer (3q21) to MECOM (3q26) results in overexpression of the MECOM isoform EVI1 and monoallelic expression of GATA2 from the unaffected allele. The full-length MECOM transcript, MDS1-EVI1, is not expressed as the result of the 3q26 rearrangement. Besides the classical inv(3)/t(3;3), a number of other 3q26/MECOM rearrangements with poor treatment response have been reported in AML. Here we demonstrate, in a group of 33 AML patients with atypical 3q26 rearrangements, MECOM involvement with EVI1 overexpression, but no or low MDS1-EVI1 levels. Moreover, the 3q26 translocations in these AML patients often involve super-enhancers of genes active in myeloid development (e.g. CD164, PROM1, CDK6 or MYC). In more than 50% of these cases allele specific GATA2 expression was observed, either by copy number loss or by an unexplained allelic imbalance. Altogether, atypical 3q26 recapitulate the main leukemic mechanism of inv(3)/t(3;3) AML, namely EVI1 overexpression driven by enhancer hijacking, absent MDS1-EVI1 expression and potential GATA2 involvement. Therefore, we conclude that both atypical 3q26/MECOM and inv(3)/t(3;3) can be classified as a single entity of 3q26-rearranged AMLs. Routine analyses determining MECOM rearrangements, EVI1 and MDS1-EVI1 expression are required to recognize 3q-rearranged AML cases.

  Study EGAS00001004325

• Partner-independent fusion gene detection by multiplexed CRISPR/Cas9 enrichment and long-read Nanopore sequencing

  Fusion genes are hallmarks of various cancer types and important determinants for diagnosis, prognosis and treatment possibilities. The promiscuity of fusion genes with respect to partner choice and exact breakpoint-positions restricts their detection in the diagnostic setting, even for known and recurrent fusion gene configurations. To accurately identify these gene fusions in an unbiased manner, we developed FUDGE: a FUsion gene Detection assay from Gene Enrichment. FUDGE couples target-selected and strand-specific CRISPR/Cas9 activity for enrichment and detection of fusion gene drivers (e.g. BRAF, EWSR1, KMT2A/MLL) - without prior knowledge of fusion partner or breakpoint-location - to long-read Nanopore sequencing. FUDGE encompasses a dedicated bioinformatics approach (NanoFG) to detect fusion genes from Nanopore sequencing data. Our strategy is flexible with respect to target choice and enables multiplexed enrichment for simultaneous analysis of several genes in multiple samples in a single sequencing run. We observe on average a 508-fold on-target enrichment and identify fusion breakpoints at nucleotide resolution - all within two days. We demonstrate that FUDGE effectively identifies fusion genes in cancer cell lines, tumor samples and on whole genome amplified DNA irrespective of partner gene or breakpoint-position in 100% of cases. Furthermore, we show that FUDGE is superior to routine diagnostic methods for fusion gene detection. In summary, we have developed a rapid and versatile fusion gene detection assay, providing an unparalleled opportunity for pan-cancer detection of fusion genes in routine diagnostics.

  Study EGAS00001003964

• Whole transcriptome profiling of liquid biopsies from tumour xenografted mouse models: validation cohort

  While cell-free DNA (cfDNA) in liquid biopsies is widely being used and investigated, free circulating RNA (extracellular RNA, exRNA) has the potential to improve therapy response monitoring and cancer detection due to its dynamic nature. A fundamental open question hampering the initiation of large-scale liquid biopsy collections for tumour exRNA analysis is that it remains unclear in which blood subcompartment tumour-derived exRNAs primarily reside. We set out to develop a host-xenograft deconvolution framework, exRNAxeno, with cDNA mapping strategies to either a combined human-mouse reference genome or both species genomes in parallel, that can be applied to exRNA sequencing data from liquid biopsies of human xenograft mouse models, enabling to distinguish (human) tumoural RNA from (murine) host RNA, and as such to specifically analyse tumour-derived exRNA. Subsequently, the preferred exRNAxeno combination pipeline was applied to total exRNA sequencing data from blood platelets and three plasma fractions from a breast cancer patient-derived and neuroblastoma cell line-derived xenograft mouse model. We show that tumoural exRNA concentrations are not determined by plasma platelet levels, while host exRNA concentrations increase with platelet content. Furthermore, a large variability in exRNA levels and gene content across individual mice is observed. In general, the tumoural gene detectability in plasma is correlated with the RNA expression levels in the tumour tissue or cancer cell line. Our results unravel new aspects of tumour-derived exRNA biology in xenograft mouse models.

  Study EGAS00001006582

• Differentially methylated CpGs in response to growth hormone administration in children with idiopathic short stature

  Background: Recombinant human growth hormone (rhGH) has shown a great growth-promoting potential in children with idiopathic short stature (ISS). However, the response to rhGH differs across individuals, largely due to genetic and epigenetic heterogeneity. Since epigenetic marks on the methylome can be dynamically influenced by GH, we performed a comprehensive pharmaco-epigenomics analysis of DNA methylation changes associated with long-term rhGH administration in children with ISS. Results: We measured DNA methylation profiles before and after GH treatment (with a duration of ~18 months in average) on 47 healthy children using customized methylC-seq capture sequencing. Their changes were compared and associated with changes in plasma IGF1 by adjusting sex, age, treatment duration, and estimated blood proportions. We observed a considerable inter-individual heterogeneity of DNA methylation changes responding to GH treatment. We identified 267 response-associated differentially methylated cytosines (DMC) that were enriched in promoter regions, CpG islands and blood cell-type specific regulatory elements. Furthermore, the genes associated with these DMCs were enriched in the biology process of "cell development", "neuron differentiation" and "developmental growth", and in the TGF-beta signaling pathway, PPAR Alpha pathway, endoderm differentiation pathway, adipocytokine signaling pathway as well as PI3K-Akt signaling pathway, and cAMP signaling pathway. Conclusion: Our study provides a first insight in DNA methylation changes associated with rhGH administration, which may help understand mechanisms of epigenetic regulation on GH-responsive genes.

  Study EGAS00001006115

• Circulating cell-free DNA methylation profiles as noninvasive multiple sclerosis biomarkers: A proof-of-concept study

  In chronic neurodegenerative diseases such as multiple sclerosis (MS), there is a critical unmet need for cost-effective non-invasive blood biomarkers to concurrently classify disease subtypes, monitor disability severity, and predict long-term progression. In this proof-of-concept study, we performed low-coverage whole-genome bisulfite sequencing (WGBS) on 75 plasma cell-free DNA (cfDNA) samples collected from a well-characterized real-world prospective clinic cohort with longitudinal disability outcome measurements. We assessed the clinical utility of cfDNA methylation profile for differentiating MS patients from non-MS controls, classifying MS subtypes, estimating disability severity, and predicting disease trajectories.

  Study EGAS50000001277

• GCAT | Genomes for life

  The GCAT project is a prospective cohort study that was designed to recruit the general population of Catalonia, the north-east region of Spain. This study is a long-term genomic, environmental and lifestyle cohort project that aims to evaluate and track multiple pathologies as well as biologically related traits. By the end of 2017, the GCAT Study had recruited a total of 20 000 participants aged 40–65 years. Participants who agreed to take part in the study completed a self-administered computer-driven questionnaire, and underwent blood pressure, cardiac frequency and anthropometry measurements. For each participant, blood plasma, blood serum and white blood cells are collected at baseline. The GCAT Study has access to the electronic health records of the Catalan Public Healthcare System. Participants are followed biannually for/during, at least, 20 years after recruitment. Therefore, the GCAT Study offers a unique opportunity to integrate diverse data to allow the identification of novel relations among different biomarkers and conditions. Results may lead to the development of new genetic, genomic, epigenomic and proteomic diagnoses and screening tests, as well as new public health recommendations. The only currently available population-based prospective cohort study in Spain to the date, including more than 4899 genotyped participants and 800 whole genome sequences is now stored in the EGA archive under accession id EGAS00001003018.

  Blog gcat-genomes-for-life

• RNA sequencing of a glioblastoma PDX cohort

  Patient-derived xenograft (PDX) models represent an essential tool for the preclinical evaluation of novel targeted therapies and their combinations. In this project, we molecularly characterized a comprehensive panel of glioma PDX models with well conserved molecular and pathological features over multiple passages. PDX samples were subjected to next generation sequencing (NGS). Characterization of cancer-relevant features including driver mutations or cellular processes was performed using mutational and gene expression data in order to identify potential biomarker or treatment targets in glioblastoma. In summary, we reported a newly established and molecularly characterized panel of glioma PDX models with high relevance for translational preclinical research.

  Study EGAS00001007119

• MorphoITH: A Framework for Deconvolving Intra-Tumor Heterogeneity Using Tissue Morphology

  This study introduces MorphoITH, a framework for deconvolving intra-tumor heterogeneity (ITH) in clear cell renal cell carcinoma (ccRCC) based on tissue morphology from histopathological slides. The study tested MorphoITH's ability to capture different aspects of morphology and to reflect genetic ITH. The data deposited here is derived from a morphology guided multi-regional sequencing dataset used to study the relationship between morphology and genetic evolution. This dataset consists of whole exome sequencing of tumor from multiple sites and adjacent normal samples which were collected from two patients treated in affiliated hospitals of the University of Texas Southwestern Medical Center (UTSW), including Parkland Hospital.

  Study EGAS50000001064