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• Blueprint Data Access Committee.

  Access to the sequence and alignment and genotype level data produced by the Blueprint Consortium is controlled by the Blueprint Data Access Committee (DAC). Access to data will be granted to qualified investigators for appropriate use. Please email blueprint-dac@ebi.ac.uk to request the Data Access Application and Data Access Agreement.

  Dac EGAC00001000135

• DNA Methylation in Prostate Tumor and Paired Benign Tissue for African and European Ancestry Men

  We recruited prostate cancer (PCa) patients undergoing robotic-assisted laparoscopic radical prostatectomy at the University of Chicago Medical Center Urology Clinic between 2011 and 2017. Our study includes 76 African American (AA) and 75 European American (EA) men with PCa of Gleason Score (GS) ≥7, recruited by the Epidemiology Research Recruitment Core at the University of Chicago. All patients consented to the collection of questionnaire data, prostate tissue, and medical records. The study was approved by the Institutional Review Board of the University of Chicago. Following prostatectomy, prostate tissue was sent to the Human Tissue Research Center (HTRC) at the University of Chicago. Each sample underwent histological examination and Gleason scoring by University of Chicago genitourinary pathologists. The presence of adenocarcinoma was confirmed by overexpression of alpha-methylacyl-coenzyme-A racemase (AMACR) and areas for DNA extraction were marked. Tissue samples used for DNA extraction were obtained using with either a 1mm biopunch or laser microdissection of a 100 µm2 area of tissue. Genome-wide DNA methylation profiles were generated using Illumina's Infinium MethylationEPIC BeadChip array kit. Genome-wide SNP data was generated (from benign tissue DNA) using the Illumina Infinium Multi-Ethnic Global-8 v1.0 array at the University of Chicago Genomics Core Facility. We performed imputation using the Haplotype Reference Consortium (HRC, Version r1.1 2016) panel, which includes all samples from the 1,000 Genomes Project (Phase 3), using the Michigan Imputation Server. The imputed SNP data for mQTL analysis was imputed based on genotype SNP data which was all from benign tissue DNA and the methylation data was identified in both tumor and benign tissue, both AA and EA men, and identified QTLs co-occuring with prostate cancer susceptibility loci.Data including SNP genotypes, DNA methylation, tissue type, and participant characteristics will be available in dbGaP.

  Study phs003516

• Integrative analysis reveals a macrophage-predominant, immunosuppressive immune microenvironment and subtype-specific therapeutic vulnerabilities in advanced salivary gland cancer

  Background Salivary gland cancers (SGC) are rare and heterogeneous malignant tumors. Advanced SGC lack established treatment options and show poor response to immunotherapy. Here, an integrative multi-omics analysis in a large cohort of advanced SGC revealed insights into the tumor immune microenvironment (TIM) and distinct mechanisms of immune evasion. Methods A total of 104 patients with recurrent/metastatic SGC from the DKTK MASTER program were included in this study. Whole-exome or whole-genome sequencing and RNA-sequencing was performed on fresh frozen tumor tissue. The tumor immune microenvironment was analyzed using CIBERSORT deconvolution analysis and immune gene expression scores in bulk RNA-sequencing data. Single-nuclei sequencing and immunohistochemistry analyses were performed in selected samples. Results were validated in bulk RNA-sequencing data of a previously published independent dataset. Results Bulk transcriptome analysis revealed an immune-deserted TIM in the majority of advanced SGC samples. Immune exclusion was most prominent in adenoid cystic carcinoma (ACC) subgroup 1 exhibiting a downregulation of the antigen processing machinery. Only a small subset of advanced SGC, including few adenoid cystic carcinoma, exhibited T-cell inflammation, which was correlated with tumor mutational burden in Non-ACC samples. Subtype specific expression of immune checkpoints as well as cancer testis antigens were identified with prominent expression of VTCN1 in luminal cells within ACC. Single-cell RNA-sequencing and bulk RNA-seq deconvolution analysis validated immune cell exclusion and revealed a TIM that was dominated by M2 macrophages across SGC subtypes. Among evaluable patients treated with immune checkpoint inhibitors, a high T-cell to macrophage ratio was associated with clinical benefit. Conclusions These data support biomarker-based development of immune-checkpoint inhibition and the development of novel immune-checkpoint inhibitors and cellular therapies in SGC.

  Study EGAS50000000809

• Na?ve Treg-specific genomic DNA hypomethylation for autoimmune disease susceptibility

  Naturally occurring regulatory T (Treg) cells, which are specifically expressing the transcription factor FOXP3, CD25 and CTLA-4, are indispensable for the maintenance of immunological self-tolerance and homeostasis. Mutations of the FOXP3, CD25 and CTLA4 genes compromise the function of natural FOXP3+CD25+CD4+ Treg cells and cause severe autoimmune diseases showing Mendelian inheritance. Also, altered Treg-specific epigenetic changes, such as DNA hypomethylation and enhancer activation, impair the expression of these molecules in developing Treg cells, causing autoimmune diseases. It is obscure, however, whether Treg cells significantly contribute to the development of polygenic common autoimmune diseases (such as type 1 diabetes and rheumatoid arthritis), which afflict ~10% of the population. Recent genome-wide association study (GWAS) of common autoimmune diseases has revealed many autoimmune disease-associated single nucleotide polymorphisms (SNPs) in enhancer regions of immune cells, for example, in non-coding regions of T cell activation-associated genes such as CD25 and CTLA4. These findings, especially the involvement of common T-cell genes in the functions of both Treg cells and effector T cells, prompt one to ask whether autoimmune disease-associated causative SNPs impact on the functions of either disease-causing effector T cells or disease-suppressing Treg cells. Here, we have explored epigenetic profiles of human Treg and conventional T (Tconv) cells in na?ve or activated states, and found that autoimmune disease-associated SNPs are predominantly enriched in Treg-specific CpG demethylated regions (Treg-DRs), which were mostly included in Treg-specific super-enhancers. Treg-DRs are developmentally established in na?ve Treg cells before antigenic stimulation, distinct from activation-induced CpG hypomethylation in Treg or Tconv cells, and highly associated with Treg cell-specific transcriptional and epigenetic changes. Taken together, genetic variants affecting endogenous development of natural Treg cells and their Treg-intrinsic functions are causative of common autoimmune diseases, indicating natural Treg cells as a key target for their treatment and prevention.

  Study JGAS000145

• First genome-wide association study in an Australian Aboriginal population provides insights into genetic risk factors for Body Mass Index and Type 2 Diabetes

  A body mass index (BMI) >22kg/m2 is a risk factor for type 2 diabetes (T2D) in Aboriginal Australians. To identify loci associated with BMI and T2D we undertook a genome-wide association study using 1,355,133 quality-controlled single nucleotide polymorphisms (SNPs) genotyped (Illumina 2.5M Duo Beadchip) in 402 individuals in extended pedigrees from a Western Australian Aboriginal community. Imputation using the thousand genomes (1000G) reference panel extended the analysis to 6,724,284 post quality-control autosomal SNPs. No associations achieved genome-wide significance, commonly accepted as P<5x10-8. Nevertheless, genes/pathways in common with other ethnicities were identified despite the arrival of Aboriginal people in Australia >45,000 years ago. The top hit (rs10868204 Pgenotyped=1.50x10-6; rs11140653 Pimputed_1000G=2.90x10-7) for BMI lies 5’ of NTRK2, the type 2 neurotrophic tyrosine kinase receptor for brain-derived neurotrophic factor (BDNF) that regulates energy balance downstream of melanocortin-4 receptor (MC4R). PIK3C2G (rs12816270 Pgenotyped=8.06x10-6; rs10841048 Pimputed_1000G=6.28x10-7) was associated with BMI, but not with T2D as reported elsewhere. BMI also associated with CNTNAP2 (rs6960319 Pgenotyped=4.65x10-5; rs13225016 Pimputed_1000G=6.57x10-5), previously identified as the strongest gene-by-environment interaction for BMI in African-Americans. The top hit (rs11240074 Pgenotyped=5.59x10-6, Pimputed_1000G=5.73x10-6) for T2D lies 5’ of BCL9 that, along with TCFL2, promotes beta-catenin’s transcriptional activity in the WNT signaling pathway. Additional hits occurred in genes affecting pancreatic (KCNJ6, KCNA1) and/or GABA (GABRR1, KCNA1) functions. Notable associations observed for genes previously identified at genome-wide significance in other populations included MC4R (Pgenotyped=4.49x10-4) for BMI and IGF2BP2 Pimputed_1000G=2.55x10-6) for T2D. Our results may provide novel functional leads in understanding disease pathogenesis in this Australian Aboriginal population.

  Study EGAS00001001004

• Nala GSI GSAv3 PGx Study

  Background: Microarrays are a well-established and widely adopted technology capable of interrogating hundreds of thousands of loci across the human genome. Combined with imputation to cover common variants not included in the chip design, they offer a cost-effective solution for large-scale genetic studies. Beyond research applications, this technology can be applied for testing pharmacogenomics, nutrigenetics, and complex disease risk prediction. However, establishing clinical reporting workflows requires a thorough evaluation of the assay's performance, which is achieved through validation studies. In this study, we performed pre-clinical validation of a genetic testing workflow based on the Illumina Global Screening Array for 25 pharmacogenomic-related genes. Methods: To evaluate the accuracy of our workflow, we conducted multiple pre-clinical validation studies. Here, we present the results of accuracy and precision assessments, involving a total of 73 cell lines. These assessments encompass reference materials from the Genome-In-A-Bottle (GIAB), the Genetic Testing Reference Material Coordination Program (GeT-RM) projects, as well as additional samples from the 1000 Genomes project (1KGP). We conducted an accuracy assessment of genotype calls for target loci in each indication against established truth sets. Results: In our per-sample analysis, we observed a mean analytical sensitivity of 99.39% and specificity 99.98%. We further assessed the accuracy of star-allele calls by relying on established diplotypes in the GeT-RM catalogue or calls made based on 1KGP genotyping. On average, we detected a diplotype concordance rate of 96.47% across 14 pharmacogenomic-related genes with star allele-calls. Lastly, we evaluated the reproducibility of our findings across replicates and observed 99.48% diplotype and 100 % phenotype inter-run concordance. Conclusion: Our comprehensive validation study demonstrates the robustness and reliability of the developed workflow, supporting its readiness for further development for applied testing.

  Study EGAS00001007710

• Copy number profiling of primary samples and cell lines of retinoblastoma

  While RB1 loss initiates retinoblastoma development, additional somatic copy number alterations (SCNAs) can drive tumor progression. Although SCNAs have been identified with good concordance between studies at a cytoband resolution, accurate identification of single genes for all recurrent SCNAs is still challenging. This study presents a comprehensive meta-analysis of genome-wide SCNAs integrated with gene expression profiling data, narrowing down the list of plausible retinoblastoma driver genes. We performed SCNA profiling of 45 primary retinoblastoma samples and 8 retinoblastoma cell lines by high-resolution microarrays. We combined our data with genomic, clinical and histopathological data of ten published genome-wide SCNA studies, which strongly enhanced the power of our analyses (N=310). Comprehensive recurrence analysis of SCNAs in all studies integrated with gene expression data allowed us to reduce candidate gene lists for 1q, 2p, 6p, 7q and 13q to a limited gene set. Besides the well-established driver genes RB1 (13q-loss) and MYCN (2p-gain) we identified CRB1 and NEK7 (1q-gain), SOX4 (6p-gain) and NUP205 (7q-gain) as novel retinoblastoma driver candidates. Depending on the sample subset and algorithms used, alternative candidates were identified including MIR181 (1q-gain) and DEK (6p gain). Remarkably, our study showed that copy number gains rarely exceeded change of one copy, even in pure tumor samples with 100% homozygosity at the RB1 locus (N=34), which is indicative for intra-tumor heterogeneity. In addition, profound between-tumor variability was observed that was associated with age at diagnosis and differentiation grades. Since focal alterations at commonly altered chromosome regions were rare except for 2p24.3 (MYCN), further functional validation of the oncogenic potential of the described candidate genes is now required. For further investigations, our study provides a refined and revised set of candidate retinoblastoma driver genes.

  Study EGAS00001001715

• The G2 gene expression signature and MYC overexpression are independent poor prognostic factors in childhood high-grade osteosarcoma

  Purpose In high-grade osteosarcoma, prognostic factors at diagnosis are insufficient for stratifying patients into relevant subgroups. Recently, a transcriptomic study developed the G1/G2 gene expression signature, in which the G2 signature was associated with unfavorable survival. An orthogonal study identified MYC amplification as an unfavorable prognostic factor using targeted next-generation sequencing. The purpose of this study was to validate the independent prognostic value and to investigate the combined prognostic value of the G1/G2 signature with, MYC amplification and/or MYC expression for survival prediction. Material and methods This study included pediatric and adolescent patients with high-grade osteosarcoma. RNA sequencing was performed in 48 patients. Whole-exome sequencing was performed in 40 patients. Gene expression signature scores, MYC amplification (defined as > 7 copies) and MYC expression levels were calculated. Multivariable Cox proportional hazards analysis was performed for event-free survival (EFS) (primary endpoint) and overall survival (OS) (secondary endpoint). Results In the full cohort, the 3-year event-free survival rate was 37%. In multivariable Cox regression analysis with metastatic disease stage (n=21, 44%) as covariate, the G2 signature and MYC expression were independently associated with worse outcomes in terms of EFS (Hazard ratio (HR) 3.32, 95% confidence interval (CI) 1.34-8.21 and HR 3.38, 95%-CI 1.71-6.66, respectively) and OS (HR 4.07, 95%-CI 1.19-13.9 and HR 2.88, 95%-CI 1.22-6.76, respectively). MYC amplification was not associated with EFS or OS in univariable analysis (HR 1.88, 95% -CI 0.74-4.77 and HR 0.79, 95%-CI 0.21-3.05, respectively). Conclusion The G2 gene expression signature and MYC expression were independently associated with unfavorable outcomes in a pediatric cohort of patients with high-grade osteosarcoma. The combined prognostic value warrants further prospective validation and could potentially serve as a stratification marker for future osteosarcoma treatment protocols.

  Study EGAS00001008073

• Single Cell Dissection of the Tumour Microenvironment Reveals Dynamic Interplay Shaping the Tumour Immunity Continuum in Ovarian Cancer

  Diverse tumour immune phenotypes with distinct T cell infiltration patterns result in different responses to cancer immunotherapies. However, the key determinants and biology underpinning these phenotypes remain elusive. Here, we provide a high-resolution dissection of the tumour microenvironment representative of different tumour immune phenotypes. By performing single-cell RNA sequencing (scRNAseq) of the tumour, immune, and stromal compartments from 15 ovarian cancer patients, we characterized the diverse cellular and functional phenotypes, as well as dynamic interplay within and between these components. Infiltrated and excluded tumours markedly differed from desert tumours in several tumour cell intrinsic features, including high interferon response, antigen presentation and oxidative phosphorylation. Moreover, these tumours were characterized by tumour associated macrophage (TAM)-like myeloid subsets, while desert tumours were enriched in myeloid derived suppressive cell (MDSC)-like myeloid subsets. Infiltrated and excluded tumours differed in their T cell composition and fibroblast subsets. While dysfunctional CD8+ GZMB T cells and IL1-activated fibroblasts (IL1 CAFs) were associated with infiltrated tumours, pre-dysfunctional CD8+ GZMK T cells and TGFB-activated fibroblasts (TGFB CAFs) were enriched in excluded tumours. These findings validated in bulk transcriptomic profiles of 1071 ovarian tumours and using in situ hybridization. Furthermore, our study revealed chemokine receptor-ligand interactions within and across compartments as potential mechanisms mediating immune cell infiltration, exemplified by the tumour cell-T cell crosstalk via a CXC16-CXCR6 axis, stromal-immune cell crosstalk via CXCL12/14-CXCR4 signalling, and TAM-like macrophage-T cell crosstalk through CXCR3-CXCL9/10/11 signalling. Our in-depth characterization of the tumour environment and intercellular interactions provides novel insights into potential molecular mechanisms that shape the distinct biology of tumour immune phenotypes. Our findings may inform novel therapeutic strategies for potentially shifting tumours along the immunity continuum to improve clinical benefit from cancer immunotherapies.

  Study EGAS00001004935

• Proteomic Analysis of Non-Muscle Invasive and Muscle Invasive Bladder Cancer Highlights Distinct Subgroups With Metabolic, Matrisomal, and Immune Hallmarks

  We present the proteome biology of bladder cancer based on a cohort comprising treatment-naïve non-muscle invasive (NMIBC, N=17) and treatment-naïve muscle-invasive bladder cancer (MIBC, N=51), as well as neoadjuvant-treated MIBC (N=11); using formalin-fixed, paraffin-embedded samples and data-independent acquisition yielding >8,000 protein identifications. The NMIBC proteome reveals two subgroups that represent cell adhesion or lipid metabolism biology. MIBC, compared to NMIBC, shows an altered metabolic proteome together with enrichment of extracellular matrix (ECM), immune response, and complement proteins; in line with three proteomic MIBC subgroups that represent ECM, metabolic, or immune signatures. The ECM and immune subgroups are enriched for markers of M2-like tumor-associated macrophages and dendritic cells, while markers for natural killer cells are exclusive for the ECM subgroup, and markers for cytotoxic T-cells are a hallmark of the immune subgroup. Infiltration of CD8+ PD1+ positive cytotoxic T cells correlated with PD-L1 expression of MIBC, as demonstrated by multiplex immunofluorescence analysis. The metabolic subgroup presents a comparably immune-depleted microenvironment. YAP1 signaling is particularly prominent in the ECM subgroup. The protein BSCL2 emerges as a prognostic marker for MIBC. We highlight increased endogenous proteolysis in MIBC alongside the upregulation of matrix metalloproteases exceeding the upregulation of their endogenous inhibitors. Genomic panel sequencing underlines prevalent FGFR3 mutations in NMIBC and prevalent TP53 mutations in MIBC, but a genomic underpinning of the MIBC subgroups remains elusive. Proteogenomic analysis yields protein-level evidence for more than 1,300 single amino acid variants. The proteomes of neoadjuvant-treated MIBC resembled treatment-naïve MIBC. Tumor-stroma interaction of MIBC was further investigated by proteomic analysis of patient-derived xenografts of MIBC. Our study adds to the proteome-level understanding of bladder cancer.

  Study EGAS00001007290

• Substantial somatic genomic variation and selection for BCOR mutations in human induced pluripotent stem cells

  Human Induced Pluripotent Stem Cells (hiPSC) are an established patient-specific model system where opportunities are emerging for cell-based therapies. We compared and contrasted hiPSCs derived from different tissues, skin and blood, in the same individual. We show extensive single-nucleotide mutagenesis in all hiPSC lines, although fibroblast-derived hiPSCs (F-hiPSCs) are particularly heavily mutagenized by ultraviolet (UV)-related damage. We utilized genome sequencing data on 454 F-hiPSCs and 44 blood-derived hiPSCs (B-hiPSCs) to gain further insights. Across 324 whole genome sequenced (WGS) F-hiPSCs derived by the Human Induced Pluripotent Stem Cell Initiative (HipSci), UV-related damage is present in ~72% of cell lines, sometimes causing substantial mutagenesis (range 0.25-15 per Mb). Furthermore, we find remarkable genomic heterogeneity between independent F-hiPSC clones derived from the same reprogramming process in the same donor, due to oligoclonal populations within fibroblasts. Combining WGS and exome-sequencing data of 452 HipSci F-hiPSCs, we identify 272 predicted pathogenic mutations in cancer-related genes, of which 21 genes were hit recurrently three or more times, involving 77 (17%) lines. Notably, 151 of 272 mutations were present in starting fibroblast populations suggesting that more than half of putative driver events in F-hiPSCs were acquired in vivo. In contrast, B-hiPSCs reprogrammed from erythroblasts show lower levels of genome-wide mutations (range 0.28-1.4 per Mb), no UV damage, but a strikingly high prevalence of acquired BCOR mutations in ~57% of lines, indicative of strong selection pressure. All hiPSCs had otherwise stable, diploid genomes on karyotypic pre-screening, highlighting how copy-number-based approaches do not have the required resolution to detect widespread nucleotide mutagenesis. This work strongly suggests that models for cell-based therapies require detailed nucleotide-resolution characterization prior to clinical application.

  Dataset EGAD00001007029

• RNAseq dataset for MALT1 in MCL

  Manuscript Title: Co-targeting of BTK and MALT1 overcomes resistance to BTK inhibitors in mantle cell lymphoma Journal: Journal of Clinical Investigation Authors Vivian Changying Jiang1, Yang Liu1, Junwei Lian1, Shengjian Huang1, Alexa Jordan1, Qingsong Cai1, Fangfang Yan3, Joseph Mitchell McIntosh1, Yijing Li1, Yuxuan Che1, Zhihong Chen1, Jovanny Vargas1, Maria Badillo1, JohnNelson Bigcal1, Heng-Huan Lee1, Wei Wang1, Yixin Yao1, Lei Nie1, Christopher Flowers1, and Michael Wang1, 2* Abstract Bruton’s tyrosine kinase (BTK) is a proven target in mantle cell lymphoma (MCL), an aggressive subtype of non-Hodgkin lymphoma. However, resistance to BTK inhibitors is a major clinical challenge. We here report that MALT1 is one of the top overexpressed genes in ibrutinib-resistant MCL cells, while expression of CARD11, which is upstream of MALT1, is decreased. MALT1 genetic knockout or inhibition produced dramatic defects in MCL cell growth regardless of ibrutinib sensitivity. Conversely, CARD11 knockout cells showed anti-tumor effects only in ibrutinib-sensitive cells, suggesting that MALT1 overexpression could drive ibrutinib resistance via bypassing BTK-CARD11 signaling. Additionally, BTK knockdown and MALT1 knockout markedly impaired MCL tumor migration and dissemination, and MALT1 pharmacological inhibition decreased MCL cell viability, adhesion, and migration by suppressing NF-κB, PI3K-ATK-mTOR, and integrin signaling. Importantly, co-targeting MALT1 with safimaltib and BTK with pirtobrutinib induced potent anti-MCL activity in ibrutinib-resistant MCL cell lines and patient-derived xenografts. Therefore, we conclude that MALT1 overexpression associates with resistance to BTK inhibitors in MCL, targeting abnormal MALT1 activity could be a promising therapeutic strategy to overcome BTK inhibitor resistance, and co-targeting of MALT1 and BTK should improve MCL treatment efficacy and durability as well as patient outcomes. Dataset description: The bulk RNA-seq dataset was generated for the cell lines below and used for two major purposes: 1. DEG analysis and GSEA analysis comparing IBN-R and IBN-S cells 2. DEG analysis and GSEA analysis comparing MCL cells with/without MI-2 treatment. sample Cell MI-2 Ibrutinib (IBN) Venetoclax (VEN) Used for IBN-R vs IBN-S comparison Used for MI-2 vs untreated (DMSO) H9 Granta519 - R S yes H21 Granta519 - R S yes H33 Granta519 - R S yes H10 Granta519-VEN-R - R R yes H22 Granta519-VEN-R - R R yes H34 Granta519-VEN-R - R R yes H3 JeKo BTK KD_1 - R R yes yes H15 JeKo BTK KD_1 - R R yes yes H27 JeKo BTK KD_1 - R R yes yes H5 JeKo BTK KD_2 - R R yes yes H17 JeKo BTK KD_2 - R R yes yes H29 JeKo BTK KD_2 - R R yes yes H1 JeKo-1 - S R yes yes H13 JeKo-1 - S R yes yes H25 JeKo-1 - S R yes yes H7 Mino - S S yes H19 Mino - S S yes H31 Mino - S S yes H8 Mino-VEN-R - S R yes H20 Mino-VEN-R - S R yes H32 Mino-VEN-R - S R yes H11 Rec-1 - S S yes H23 Rec-1 - S S yes H12 Rec-VEN-R - S S yes H24 Rec-VEN-R - S R yes H36 Rec-VEN-R - S R yes H35 Rec-1 -- S R yes H4 JeKo BTK KD_1 + MI-2 + yes H16 JeKo BTK KD_1 + MI-2 + yes H28 JeKo BTK KD_1 + MI-2 + yes H6 JeKo BTK KD_2 + MI-2 + yes H18 JeKo BTK KD_2 + MI-2 + yes H30 JeKo BTK KD_2 + MI-2 + yes H2 JeKo-1 + MI-2 + yes H14 JeKo-1 + MI-2 + yes H26 JeKo-1 + MI-2 + yes

  Dataset EGAD00001009771

• Loss of LGR4/GPR48 causes severe neonatal salt-wasting due to disrupted WNT signaling altering adrenal zonation

  Disorders of isolated mineralocorticoid deficiency causing potentially life-threatening salt-wasting crisis early in life have been associated with gene variants of aldosterone biosynthesis or resistance, but in some patients no such variants are found. WNT/β-catenin signaling is crucial for differentiation and maintenance of the aldosterone producing adrenal zona glomerulosa (zG). We describe a highly consanguineous family with multiple perinatal deaths or infants presenting at birth with failure to thrive, severe salt-wasting crises associated with isolated hypoaldosteronism, nail anomalies, short stature, and deafness. Whole exome sequencing revealed a homozygous splice variant in the R-SPONDIN receptor LGR4 gene (c.618-1G>C) regulating WNT signaling. The resulting transcripts affected protein function and stability, and resulted in loss of Wnt/β-catenin signaling in vitro. The impact of LGR4 inactivation was analyzed by adrenal cortex specific ablation of Lgr4, using Lgr4Flox/Flox mated with Sf1:Cre mice. Inactivation of Lgr4 within the adrenal cortex in the mouse model caused decreased WNT signaling, aberrant zonation with deficient zG and reduced aldosterone production. Thus, human LGR4 mutations establish a direct link between LGR4 inactivation and decreased canonical WNT signaling with abnormal zG differentiation and endocrine function. Therefore, variants in WNT signaling and its regulators should systematically be considered in familial hyperreninemic hypoaldosteronism.

  Study EGAS00001006808

• Single cell transcriptional characterization of human megakaryocyte lineage commitment and maturation

  Single cell RNA-sequencing of sternal bone marrow reciding Hematopoietic Stem Cells (HSCs) and Megakaryocytes (MKs) from individuals undergoing elective open heart valve replacement. HSCs were defined as Lineage-, CD34+, CD38-, CD45RA-, CD90+, CD49f+ cells. MKs where CD41a+, CD42b+ and ploidy was determined with Hoechst. A sternal bone marrow scraping was taken directly following median sternotomy using a Volkmann’s spoon. The sample was collected into an EDTA Vacutainer tube containing 1.8mg/ml EDTA. 4mL of Dulbecco’s phosphate buffered saline (PBS, Sigma) containing 10% human serum albumin (HSA, Gemini Bio Products) was added and the whole volume was resuspended by pipetting 2-3 times. The sample was then put on metallic thermal beads (ThermoFisher Scientific) at a temperature between 0-4°C and transported to the University of Cambridge for further processing. For HSC isolation the cells were stained with the following antibody cocktail: PECy5 conjugated anti-lineage specific antibodies: CD2 (BD), CD3 (BD), CD10 (BD), CD11b (BD), CD11c (BD), CD19 (BD), CD20 (BD), CD56 (BD), biotinylated CD42b (Pab5, NHS Blood and Transplant, International Blood Group Reference Laboratory [IBGRL]), biotinylated GP6 (Pab5, NHS Blood and Transplant, International Blood Group Reference Laboratory [IBGRL]) used in combination with PECy5 conjugated streptavidin (Biolegend). Alexa Fluor 700 conjugated anti-CD34 (BD), PerCP-Cy5.5 conjugated anti-CD38 (BD), Pacific Blue conjugated anti-CD45RA (Invitrogen), PECy7 conjugated anti-CD90 (BD),PE conjugated anti-CD49f (BD). After staining cells were kept at 4°C before sorting using a FACS Aria Fusion flow sorter (BD). Single HSCs defined as Lineage-, CD34+, CD38-, CD45RA-, CD90+, CD49f+ cells were sorted by FACS directly into individual wells of a 96-well plate. Index sort data was collected for each single cell. For MK isolation the cells were stained for surface MK markers with mouse anti-human CD41a APC conjugated antibody (BD) and mouse anti-human CD42b PE conjugated antibody (BD) and for ploidy analysis with 1ug/ml Hoechst 33342 (Invitrogen). After incubation at 37°C for 30 minutes, the cells were kept at 4°C before sorting using a FACS Aria Fusion flow sorter (BD). Single cells and MK pools of 20 cells were sorted by FACS according to ploidy level using a 100uM nozzle directly into individual wells of a 96-well plate. cDNA synthesis and poly(A) enrichment was performed following the G&T-seq protocol (Macaulay et al. 2015), a variation of the Smart-seq2 protocol1. ERCC spike-in RNA (Ambion) was added to the lysis buffer in a dilution of 1:4,000,000.

  Dataset EGAD00001006677

• Whole Genome Sequencing of clonal expansions of single healthy somatic cells (human, female)

  HSC73_clone: Bone marrow mononuclear cells from the healthy 73 years old female were thawed and labeled with Alexa-Fluor 488-conjugated anti-CD34 (581, Biolegend), Alexa-Fluor 700-conjugated anti-CD38 (HIT2, eBioscience), a cocktail of APC-conjugated lineage antibodies consisting of anti-CD4 (RPA-T4), anti-CD8 (RPA-T8), anti-CD11b (ICRF44), anti-CD20 (2H7), anti-CD56 (B159, all BD Biosciences), anti-CD14 (61D3), anti-CD19 (HIB19) and anti-CD235a (HIR2, all eBiocience) and 1 micro-gram/ml propidium iodide (Sigma). Using a BD FACSAria cell sorter, single Lin-CD34+CD38-PI- cells were individually sorted into low-adhesion 96-well tissue culture plates (Corning) containing 100micro-litre of StemSpan Serum-Free Expansion Medium (Stemcell technologies) supplemented with 100ng/ml of human SCF and FLT-3L, 50ng/ml of human TPO, 20ng/ml of human IL-3, IL-6 and G-CSF (all cytokines from Peprotech) and 50U/ml of penicillin and 50μg/ml of streptomycin (Sigma). Cells were incubated at 37 degrees C in a humidified atmosphere with 5% CO2 in air. After 5 days in culture, another 100micro litres of cytokine-containing medium were added. 13 days after seeding, clones B6 and G2 had expanded to approx. 105 cells and were selected for whole genome sequencing (2x101bp, paired-end, Illumina HiSeq2500) after tagmentation-based library preparation (see Extended Experimental Procedures) for clone B6 and standard library preparation for clone G2. For germline-control ~106 unsorted bone marrow mononuclear cells from the same donor were used for sequencing. An average of 30-fold sequence coverage for each the clones and the matching control were obtained.L4clone: A progenitor cell clone was raised from a peripheral blood sample of the 39 year old healthy female. Frozen peripheral blood mononuclear cells (PBMCs) were isolated from 2 ml heparinised peripheral blood via Ficoll Paque density centrifugation. A methylcellulose assay was performed as described earlier (Weisse et al., 2012). In brief, non-adherent mononuclear cells were incubated in the presence of the recombinant human cytokines IL-3, IL-5 and GM-CSF (R&D systems) over 14 days to induce colony formation. Colonies were detected under an inverted light microscope, and plucked by a pipette when colonies had approximately 10,000 cells/CFU. Each colony was washed three times in PBS and finally frozen as a cell pellet in -80 degrees C. Genomic DNA was isolated using the QIAamp DNA micro kit according to the instructions of the manufacturer (Qiagen, Hilden, Germany). Whole genome sequencing (2x101bp, paired-end, Illumina HiSeq2500) was performed for colony 4 after tagmentation-based library preparation and resulted in 15-fold sequence coverage for each the colony and the matching whole blood.

  Dataset EGAD00001000666

• Genomic Analysis of Low Grade B-Cell Lymphoma

  We included tumors from 64 newly diagnosed or untreated Low Grade B-Cell Lymphoma (LGBCL) patients, consisting of SMZL (n = 48), NMZL (n = 6), EMZL (n = 2), LPL (n = 5), and B-NOS (n = 3) in this study. Samples were selected from cases consented to the Molecular Epidemiology Resource (MER) of the University of Iowa and Mayo Clinic Lymphoma Specialized Program of Research Excellence (SPORE). RNA or DNA were extracted from 64 frozen LGBCLs tumors (with available matched germline DNA for N=61. For RNA sequencing, library preparation was done using the Illumina TruSeq RNA Exome Kit and sequenced with 100 nucleotide paired-end reads using the HiSeq 4000. For WES, library preparation was done using the Agilent SureSelect XT kit and sequenced with 100 nucleotide paired-end reads using the Illumina HiSeq 4000.

  Study phs002552

• National Cancer Institute Genome-Wide Association Study of Renal Cell Carcinoma

  The National Cancer Institute (NCI) genome-wide association study (GWAS) of renal cell carcinoma (RCC) was conducted to investigate common genetic variants associated with RCC risk. The GWAS includes 1,453 RCC cases and 3,531 controls of European background from 4 studies (3 cohort, 1 case-control), scanned using the Illumina InfiniumHumanHap 550, 610 and 660W chips. This project was supported by the Intramural Research Program of the National Institutes of Health and NCI. Data from this GWAS were pooled with those from another GWAS of RCC (2,639 cases and 5,392 controls) conducted in Europe by the International Agency for Research on Cancer and the Centre National de Gènotypage. Findings from this collaboration are described in an upcoming report (Purdue et al. Nature Genetics 2011;43(1):60-65; PMID: 21131975). Only data from the NCI scan are included in this dbGaP submission.

  Study phs000351

• Deciphering Maternal-Fetal Crosstalk in the Human Placenta During Parturition Using Single-Cell RNA-Sequencing

  Labor is a complex physiological process requiring a well-orchestrated dialogue between the mother and fetus. However, the cellular contributions and communications that facilitate maternal-fetal crosstalk in labor have not been fully elucidated. In this latest version (#5), scRNA-seq was applied to decipher maternal-fetal signaling in the human placenta and chorioamniotic membranes during term labor. Samples were obtained from women who underwent spontaneous labor (n = 24) or who were delivered in the absence of spontaneous labor (n = 18) at term. Samples from previous versions (#1-3) of this study include the placental villous tree, basal plate, and chorioamniotic membranes of women across a range of pregnancy physiologic and pathologic conditions, including the processes of labor (term labor and preterm labor), and COVID-19 (version #3). Version #4 includes samples from the human myometrium during spontaneous term labor.

  Study phs001886

• NHLBI TOPMed - NHGRI CCDG: Early-onset Atrial Fibrillation in the CATHeterization GENetics (CATHGEN) Cohort

  The CATHeterization GENetics (CATHGEN) biorepository collected biospecimens and clinical data on individuals age ≥18 undergoing cardiac catheterization for concern of ischemic heart disease at a single center (Duke University Medical Center) from 2000-2010; a total of N=9334 individuals were collected. Samples were matched at the individual level to clinical data collected at the time of catheterization and stored in the Duke Databank for Cardiovascular Diseases (DDCD). Clinical data included subject demographics, cardiometabolic risk factors, cardiac history including symptoms, age-of-onset of cardiovascular diseases, coronary anatomy and cardiac function at catheterization, laboratory data, and yearly follow-up for hospitalizations, vital status, medication use and lifestyle factors. AF cases were defined as individuals who had ever had AF based on any ECG available at Duke University or ICD-9 code for AF used for inpatient or outpatient billing.

  Study phs001600

• Dynamics of Genomic and Immune Responses During Primary Immunotherapy Resistance in Mismatch Repair Deficient Tumors

  We study a patient with dMMR lung cancer refractory to immunotherapy. Post-treatment tumor demonstrated compound heterozygous frameshift deletions located upstream of the kinase domain in the gene encoding JAK1 protein, downregulation of JAK1 and mediators of its signal transduction, and total loss of JAK1 phosphorylation. Importantly, one of the JAK1 mutations, despite not being detected in the pre-treatment tumor, was found at low variant allele frequency in the pre-treatment circulating tumor DNA, suggesting clonal selection of the mutation. This report provides the most detailed look yet of defective JAK1 signaling in the context of dMMR and immunotherapy-resistance. Together with observations of JAK1 frameshift indels being enriched in dMMR compared with MMR-proficient tumors, our findings demonstrate the critical function of JAK1 in immunological surveillance of dMMR cancer.

  Study phs002089

• Minnesota Center for Twin and Family Research (MCTFR) Genome-Wide Association Study of Behavioral Disinhibition

  This is an epidemiological study of substance abuse and related psychopathology in which the subjects have been drawn from the Minnesota Center for Twin and Family Research (MCTFR). The MCTFR is a large, ongoing, family-based study. It is in part a longitudinal study of two cohorts of adolescent twins and their parents. It additionally includes a parallel longitudinal study of adolescent adoptive siblings, biologically related siblings, and their parents. Over 1500 twin families and 350 adoptive and biological sibling families have been studied, with follow-up assessments occurring approximately every 3 years. The MCTFR gathered detailed, standardized data on study participants including DSM-IIIR and DSM-IV diagnostic interview and questionnaire data. For the Genome-Wide Association Study of Behavioral Disinhibition, parental intake data plus that adolescent data gathered closest to the children 17th birthday (between ages 16.5 and 21) was used.

  Study phs000620

• Genome-wide Association Study and Meta-Analysis of Ewing Sarcoma

  We combined a set of 122 French Ewing sarcoma (EWS) cases from the Institut Curie, 19 EWS cases from the National Cancer Institute (NCI) Center for Cancer Research (CCR), and 29 EWS cases from the NCI Bone Disease and Injury Study. All EWS cases were confirmed by medical record review and the presence of a specific EWSR1-ETS translocation were noted when data was available. Each participant provided informed consent and each participating study was approved by the Institutional Review Boards of their study center. DNA was extracted using standard methods from blood, saliva (Oragene), or buccal cells. All de novo genotyping of EWS cases was performed at the NCI CGR on the Illumina OmniExpress-24 v1.1 array. Genotyping was performed according to manufacturer's guidelines using the Infinium HD Assay automated protocol.

  Study phs001549

• Genomic Analysis of Peripheral T-Cell Lymphomas

  Peripheral T-cell lymphomas (PTCLs) are a heterogeneous and poorly understood group of non Hodgkin lymphomas. We aim to identify new genetic alterations in PTCL transformation by using a combination of whole exome sequencing of tumor-normal DNA pairs, RNAseq analysis and targeted deep sequencing of candidate genes. Our data identified highly recurrent mutations in epigenetic factors such as TET2, DNMT3A and IDH2 as well as a new highly prevalent mutation in RHOA, the G17V allele that is present in almost 70% of angioimmunoblastic T-cell lymphomas (AITL) and almost 20% of not otherwise specified PTCL (PTCL NOS) samples. In addition, we describe new and recurrent genetic defects including mutations in FYN, ATM, B2M and CD58 implicating SRC signaling, impaired DNA damage response and escape from immune surveillance processes as key players in the pathogenesis of PTCL.

  Study phs000689

• Genomic analysis of primary plasma cell leukemia reveals complex structural alterations and high risk mutational patterns

  Primary plasma cell leukemia (pPCL) is a rare and aggressive form of multiple myeloma (MM) that is characterized by poor overall survival despite advances in anti-MM therapy. The disease biology as well as molecular mechanisms that distinguish pPCL from non-pPCL MM remain poorly understood. In an attempt to identify key biological mechanisms that result in the aggressive pPCL phenotype we performed whole exome sequencing in 23 patients with newly diagnosed pPCL. The results reveal an enrichment of complex structural changes and high risk mutational patterns in pPCL that explain, at least in part, the aggressive nature of the disease. In particular pPCL patients with traditional low risk features such as translocation t(11;14) or hyperdiploidy accumulated adverse risk genetic events that could account for the poor outcome in this group.

  Study phs002022

• Collaborative Cohort of Cohorts for COVID-19 Research (C4R): REasons for Geographic and Racial Differences in Stroke (REGARDS)

  Cohort DescriptionThe REasons for Geographic and Racial Differences in Stroke (REGARDS) cohort is one of the nation's largest, most comprehensive population-based cohorts, and it uses innovative home- and telephone-based data collection. REGARDS centrally recruited and initially examined 30,239 Black and White men and women aged ≥45 years in 2003-7 to understand why Southerners and Black Americans have a higher incidence of stroke and related diseases that affect brain health.Data Being Submitted Wave 1 questionnaire data includes 397 variables for 8109 REGARDS participants in C4R. Wave 2 questionnaire data includes 448 variables for 6421 REGARDS participants in C4R. Dried Blood Spot/Serosurvey data includes 7 variables for 4058 REGARDS participants in C4R. Derived data includes 43 variables for 8606 REGARDS participants in C4R. Phenotype data includes 113 variables for 7880 REGARDS participants in C4R.

  Study phs002919