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• Singel-cell RNA sequencing and CUT&RUN sequencing of human RUNX2-deficient osteoblasts

  Runx2 is a master regulator of bone formation, and its dysfunction causes cleidocranial dysplasia (CCD) in humans. When iPS cells were generated from patients with CCD and Runx2-deficient iPS cells were generated using gene-editing techniques, abnormal laminopathy-like nuclei were observed. Runx2-deficient cells showed reduced Lamin A/C expression, but not protein levels. However, in Runx2-deficient cells, both the gene expression and protein levels of Nesprin-1 were reduced, perinuclear actin fibers were sparser, and nuclear stiffness was reduced. Forced expression of Lamin A/C increased nuclear stiffness but did not improve nuclear morphology. In contrast, the induction of Nesprin-1 expression alone normalized nuclear stiffness and restored nuclear morphology and perinuclear actin distribution. In Runx2-null cells, mechanical stress-induced phosphorylation of emerin was not observed. In contrast, forced expression of nesprin-1 in Runx2-null cells resulted in phosphorylation of emerin, indicating the restoration of intracellular tension. These observations were confirmed by atomic force microscopy. Therefore, the intracellular tension was inferred to pull the nuclear membrane into its normal shape. CUT&RUN assay and single RNA-seq analysis showed that an aberrant nuclear membrane caused loss of nuclear lamina gene regulation machinery, making the progression of normal osteogenic differentiation impossible; however, supplementation with Nesprin-1 restored gene regulation mechanisms and promoted pre-osteoblast formation with normal nuclear morphology. Nesprin-1 expression induced by Runx2 is essential for epigenetic regulation of the nuclear lamina. We propose CCD as a new type of laminopathy involving defective expression of Nesprin-1 regulated by Runx2.

  Study JGAS000663

• The genomic landscape of pediatric acute lymphoblastic leukemia

  Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer and arises from B- or T-lineage lymphocyte precursors. ALL comprises dozens of subtypes, and genomic analyses have largely been performed within these subtypes. Here we analyze pediatric ALL genomes across all subtypes, including 768 whole genomes, 1,729 exomes, and 1,889 transcriptomes in 2,754 patients. Most ALL subtypes harbor 4 or more driver alterations per sample, similar to adult cancers, despite low mutation burdens. Hyperdiploid B-ALL copy gains are likely acquired early and synchronously, with copy gains occurring before ultraviolet-induced mutations. By contrast, ultraviolet-induced mutations precede copy gains in iAMP21 B-ALL. Overall, we identified 378 putative ALL driver genes. Most driver alterations vary in prevalence across ALL subtypes, with B-ALL enriched for Ras and B-lineage-related alterations, and T-ALL enriched for PI3K, JAK, and cell cycle alterations. Most B-ALL (54.3%) and T-ALL (51.2%) samples bear at least one rare driver gene alteration (present in less than 2% of samples), including 30 putative novel cancer driver genes associated with ubiquitination, SUMOylation, non-coding, and other functions. Known or novel alterations associated with poor outcomes in specific subtypes include TBL1XR1 alterations in ETV6-RUNX1 patients, CREBBP in Ph-like-CRLF2, SETD2 in hyperdiploid, and PTEN in TAL1 patients. Intriguingly, DUX4 and KMT2A subtypes separate into CEBPA/FLT3- or NFATC4-expressing subgroups with potential clinical implications. Together, these results deepen understanding of ALL etiology and outcomes, and facilitate ALL model development.

  Study EGAS00001005250

• Exome sequencing of uterine leiomyosarcomas

  Uterine leiomyosarcomas (ULMSs) are aggressive smooth muscle tumors associated with poor clinical outcome. Despite previous cytogenetic and molecular studies, their molecular background has remained elusive. To examine somatic variation in ULMS, we performed exome sequencing on 19 tumors. Altogether 43 genes were mutated in at least two ULMSs. Most frequently mutated genes included tumor protein P53 (TP53; 6/19; 33%), alpha thalassemia/mental retardation syndrome X-linked (ATRX; 5/19; 26%), and mediator complex subunit 12 (MED12; 4/19; 21%). Unlike ATRX mutations, both TP53 and MED12 alterations have repeatedly been associated with ULMSs. All the observed ATRX alterations were either nonsense or frameshift mutations. ATRX protein levels were reliably analyzed by immunohistochemistry in altogether 44 ULMSs, and the majority of tumors (23/44; 52%) showed clearly reduced expression. Loss of ATRX expression has been associated with alternative lengthening of telomeres (ALT), and thus the telomere length was analyzed with telomere-specific fluorescence in situ hybridization. The ALT phenotype was confirmed in all ULMSs showing diminished ATRX expression. Exome data also revealed one nonsense mutation in death-domain associated protein (DAXX), another gene previously associated with ALT, and the tumor showed ALT positivity. Aberrant expression of both TP53 and ATRX were associated with poor overall survival. In conclusion, exome sequencing revealed that TP53, ATRX, and MED12 are frequently mutated in ULMSs. ALT phenotype was commonly seen in tumors, indicating that ATR inhibitors, which were recently suggested as possible new drugs for ATRX-deficient tumors, could provide a potential novel therapeutic option for ULMS.

  Study EGAS00001001612

• Aging and genome-wide patterns of DNA methylation in an African rainforest hunter-gathering population

  Aging is associated with widespread changes in genome-wide patterns of DNA methylation. Thousands of CpG sites whose tissue-specific methylation levels are strongly correlated with chronological age have been previously identified. However, the majority of these studies have focused primarily on cosmopolitan populations living in the developed world; it is not known if age-related patterns of DNA methylation at these loci are similar across a broad range of human genetic and ecological diversity. We investigated genome-wide methylation patterns using saliva derived DNA from a traditionally hunting and gathering African populations: the Baka of the western Central African rainforest, together with the ≠Khomani San of the South African Kalahari Desert. We identify hundreds of CpG sites whose methylation levels are significantly associated with age, thousands that are significant in a meta-analysis, and replicate trends previously reported in populations of non-African descent. We confirm that an age associated site in the gene ELOVL2 shows a remarkably congruent relationship with aging in humans, despite extensive genetic and environmental variation across populations. We also demonstrate that genotype state at methylation quantitative trait loci (meQTLs) can affect methylation trends at some known age-associated CpG sites. Our study explores the relationship between CpG methylation and chronological age in populations of African hunter-gatherers, who rely on different diets across diverse ecologies. While many age-related CpG sites replicate across populations, we show that considering common genetic variation at meQTLs further improves our ability to detect previously identified age associations.

  Study EGAS00001002226

• Genomic consequences of aberrant DNA repair mechanisms stratify ovarian cancer histotypes

  Aberrant DNA repair processes are a hallmark of human tumours. How these deficiencies variously impact the genomes of ovarian cancer between and within histotypes remains unknown. We studied the whole genome pointmutation and structural variation patterns of 133 tumours (59 high grade serous (HGSC), 35 clear cell (CCOC), 29 endometrioid (ENOC), and 10 adult granulosa cell tumours (GCT)) as a substrate for class discovery in ovarian cancer. Ab-initio clustering of integrated point mutation and structural variation signatures revealed seven novel subgroups, comprising between and within histotype stratification. Prevalence of fold-back inversions (FBI) co-localised with high level amplification events divided HGSC into two prognostically significant subgroups. This finding was recapitulated in two additional independent cohorts (total n=576 cases), and transcended gene based mutation status and gene expression as prognostically relevant features of HGSC. CCOC were divided into one group harbouring a signature reflective of active genome editing via APOBEC enzymes (26%) and a complementary group with an age-relatedmutational signature. ENOC were divided intomicrosatellite instable (MSI) cases (28%) with a distinctmismatch repairmutation signature and an outlying mutation rate, with the remainder of cases distributed amongst the remaining six groups. We suggest the FBI, MSI, and APOBEC groups represent biologic strata that could direct treatment strategies. Taken together, our work establishes the efficacy of the somatic genome as a biomarker to stratify ovarian cancers, simultaneously identifying patients that may benefit from emerging therapeutics and distinct subgroups within classic ovarian cancer histotypes.

  Study EGAS00001002390

• Recurrent epimutations activate gene body promoters in primary glioblastoma

  Aberrant DNA hypomethylation may play an important role in the growth rate of glioblastoma (GBM), but the functional impact on transcription remains poorly understood. We assayed the GBM methylome with MeDIP-seq and MRE-seq, adjusting for copy number differences, in a small set of non-glioma CpG island methylator phenotype (non-G-CIMP) primary tumors. Recurrent hypomethylated loci were enriched within a region of chromosome 5p15 that is specified as a cancer amplicon and also encompasses TERT, encoding telomerase reverse transcriptase, which plays a critical role in tumorigenesis. Overall, 76 gene body promoters were recurrently hypomethylated, including TERT and the oncogenes GLI3 and TP73. Recurring hypomethylation also affected previously unannotated alternative promoters, and luciferase reporter assays for three of four of these promoters confirmed strong promoter activity in GBM cells. Histone H3 lysine 4 trimethylation (H3K4me3) ChIP-seq on tissue from the GBMs uncovered peaks that coincide precisely with tumor-specific decrease of DNA methylation at 200 loci, 133 of which are in gene bodies. Detailed investigation of TP73 and TERT gene body hypomethylation demonstrated increased expression of corresponding alternate transcripts, which in TP73 encodes a truncated p73 protein with oncogenic function and in TERT encodes a putative reverse transcriptase-null protein. Our findings suggest that recurring gene body promoter hypomethylation events, along with histone H3K4 trimethylation, alter the transcriptional landscape of GBM through the activation of a limited number of normally silenced promoters within gene bodies, in at least one case leading to expression of an oncogenic protein.

  Study EGAS00001000685

• Exome Sequencing Of 75 Individuals From Multiply Affected Coeliac Families

  Coeliac disease is a highly heritable common autoimmune disease involving chronic small intestinal inflammation in response to dietary wheat. The human leukocyte antigen (HLA) region, and 40 newer regions identified by genome wide association studies (GWAS) and dense fine mapping, account for ~40% of the disease heritability. We hypothesized that in pedigrees with multiple individuals with coeliac disease rare [minor allele frequency (MAF) <0.5%] mutations of larger effect size (odds ratios of ~ 2 – 5) might exist. We sequenced the exomes of 75 coeliac individuals of European ancestry from 55 multiply affected families. We selected interesting variants and genes for further follow up using a combination of: an assessment of shared variants between related subjects, a model-free linkage test, and gene burden tests for multiple, potentially causal, variants. We next performed highly multiplexed amplicon resequencing of all RefSeq exons from 24 candidate genes selected on the basis of the exome sequencing data in 2,248 unrelated coeliac cases and 2,230 controls. 1,335 variants with a 99.9% genotyping call rate were observed in 4,478 samples, of which 939 were present in coding regions of 24 genes (Ti/Tv 2.99). 91.7% of coding variants were rare (MAF <0.5%) and 60% were novel. Gene burden tests performed on rare functional variants identified no significant associations (P<1x10-3) in the resequenced candidate genes. Our strategy of sequencing multiply affected families with deep follow up of candidate genes has not identified any new coeliac disease risk mutations.

  Study EGAS00001001093

• Cerebrospinal fluid cfDNA sequencing for classification of central nervous system glioma

  Primary central nervous system (CNS) gliomas can be classified by characteristic genetic alterations. In addition to solid tissue obtained by surgery or biopsy, cell-free DNA (cfDNA) derived from cerebrospinal fluid (CSF) is an alternative source of material for genomic analyses. Experimental design: We performed targeted next-generation sequencing (NGS) of CSF cfDNA in a representative cohort of 85 patients presenting with with suspicion of primary or recurrent glioma at two neurooncological centers. Copy-number variation (CNV) profiles, single nucleotide variants (SNVs), and small insertions/ deletions (indels) were combined into a molecular-guided tumor classification. Comparison with the solid tumor was performed for 38 cases with matching solid tissue available. Results: Cases were stratified into four groups: glioblastoma (n = 32), other glioma (n = 19), non-malignant (n = 17) and no confirmed diagnosis available (n = 17). We introduce a molecular-guided tumor classification, enabling identification of tumor entities and/ or cancer specific alterations in 75.0 % (n = 24) of glioblastoma and 52.6 % (n = 10) of other glioma cases. The overlap between CSF and matching solid tissue was highest for CNVs (26-48 %) and SNVs at pre-defined gene loci (44 %), followed by SNVs/indels identified via uninformed variant calling (8-14 %). A molecular-guided tumor classification was made 23.5 % (n = 4) of cases with no confirmed diagnosis available. Conclusions: We developed a workflow for targeted sequencing of CSF cfDNA as well as a strategy for interpretation and reporting of sequencing results based on a molecular-guided tumor classification in glioma.

  Study EGAS50000000060

• Sporadic and endemic Burkitt lymphoma have frequent FOXO1 mutations but distinct hotspots in the AKT recognition motif

  FOXO1 has an oncogenic role in adult germinal center derived lymphomas, in which mutations, predominately within the AKT recognition motif, cause nuclear retention of FOXO1 resulting in increased cell proliferation. To determine the prevalence and distribution of FOXO1 mutations in pediatric Burkitt lymphoma (BL), we sequenced a large number of sporadic and endemic BL patient samples. We report a high frequency of FOXO1 mutations in both sporadic and endemic BL at diagnosis, occurring in 23/78 (29%) and 48/89 (54%) samples respectively, as well as 8/16 (50%) cases at relapse. Mutations of T24 were the most common in sporadic BL but were rare in endemic cases, in which mutations of residue S22, also within the AKT recognition motif, were the most frequent. FOXO1 mutations were almost always present in the major tumor cell clone but were not associated with outcome. Analysis of other recurrent mutations reported in BL revealed that FOXO1 mutations were associated with mutations of DDX3X and ARID1A, but not MYC, TCF3/ID3 or members of the phosphatidylinositol 3’ OH kinase (PI3K) signaling pathway. We further show common nuclear retention of the FOXO1 protein, irrespective of mutation status, suggesting alternative unknown mechanisms for maintaining FOXO1 transcriptional activity in BL. CRISPR/Cas9 knockout of FOXO1 in an endemic cell line produced a significant decrease in cell proliferation, supporting an oncogenic role for FOXO1 in endemic BL. Thus, FOXO1 is frequently mutated in both sporadic and endemic BL and may offer a potential therapeutic target for pediatric BL patients worldwide.

  Study EGAS00001003719

• Colorectal cancer study

  Matrix metalloproteinase-11 (MMP11) is an enzyme with proteolytic activity against matrix and non-matrix proteins. Although most MMPs are secreted as inactive proenzymes and they are later activated extracellularly, MMP11 is activated intracellularly by furin within the constitutive secretory pathway. It is a key factor in physiological tissue remodeling and its alteration may play an important role in the progression of epithelial malignancies and other diseases. TCGA colon and colorectal adenocarcinoma data showed that upregulation of MMP11 expression correlates with tumorigenesis and malignancy. Here, we provide evidence that a germline variant in the MMP11 gene (NM_005940: c.232C>T; p.(Pro78Ser)), identified by whole exome sequencing, can increase the tumorigenic properties of colorectal cancer (CRC) cells. P78S is located in the prodomain region, which is responsible for blocking MMP11’s protease activity. This variant was detected in the proband and all the cancer-affected family members analyzed, while it was not detected in healthy relatives. In silico analyses predict that P78S could have an impact on the activation of the enzyme. Furthermore, our in vitro analyses show that the expression of P78S in HCT116 cells increases tumor cell invasion and proliferation. In summary, our results show that this variant could modify the structure of the MMP11 prodomain, producing a premature or uncontrolled activation of the enzyme that may contribute to an early CRC onset in these patients. The study of this gene in other CRC cases will provide further information about its role in CRC development, which might improve patient treatment in the future.

  Study EGAS00001006489