Intrinsically disordered Meningioma-1 stabilizes the BAF complex to cause AML.

Meningioma-1 (MN1) overexpression in AML is associated with poor prognosis, and forced expression of MN1 induces leukemia in mice. We sought to determine how MN1 causes AML. We found that overexpression of MN1 can be induced by translocations that result in hijacking of a downstream enhancer. Structure predictions revealed that the entire MN1 coding frame is disordered. We identified the myeloid progenitor-specific BAF complex as the key interaction partner of MN1. MN1 over-stabilizes BAF on enhancer chromatin, a function directly linked to the presence of a long polyQ-stretch within MN1. BAF over-stabilization at binding sites of transcription factors regulating a hematopoietic stem/progenitor program prevents the developmentally appropriate decommissioning of these enhancers and results in impaired myeloid differentiation and leukemia. Beyond AML, our data detail how the overexpression of a polyQ protein, in the absence of any coding sequence mutation, can be sufficient to cause malignant transformation.

A recurrent de novo splice site variant involving DNM1 exon 10a causes developmental and epileptic encephalopathy through a dominant-negative mechanism.

Heterozygous pathogenic variants in DNM1 cause developmental and epileptic encephalopathy (DEE) as a result of a dominant-negative mechanism impeding vesicular fission. Thus far, pathogenic variants in DNM1 have been studied with a canonical transcript that includes the alternatively spliced exon 10b. However, after performing RNA sequencing in 39 pediatric brain samples, we find the primary transcript expressed in the brain includes the downstream exon 10a instead. Using this information, we evaluated genotype-phenotype correlations of variants affecting exon 10a and identified a cohort of eleven previously unreported individuals. Eight individuals harbor a recurrent de novo splice site variant, c.1197-8G>A (GenBank: NM_001288739.1), which affects exon 10a and leads to DEE consistent with the classical DNM1 phenotype. We find this splice site variant leads to disease through an unexpected dominant-negative mechanism. Functional testing reveals an in-frame upstream splice acceptor causing insertion of two amino acids predicted to impair oligomerization-dependent activity. This is supported by neuropathological samples showing accumulation of enlarged synaptic vesicles adherent to the plasma membrane consistent with impaired vesicular fission. Two additional individuals with missense variants affecting exon 10a, p.Arg399Trp and p.Gly401Asp, had a similar DEE phenotype. In contrast, one individual with a missense variant affecting exon 10b, p.Pro405Leu, which is less expressed in the brain, had a correspondingly less severe presentation. Thus, we implicate variants affecting exon 10a as causing the severe DEE typically associated with DNM1-related disorders. We highlight the importance of considering relevant isoforms for disease-causing variants as well as the possibility of splice site variants acting through a dominant-negative mechanism.

Copy-number variation is an important contributor to the genetic causality of inherited retinal degenerations.

PURPOSE: Despite substantial progress in sequencing, current strategies can genetically solve only approximately 55-60% of inherited retinal degeneration (IRD) cases. This can be partially attributed to elusive mutations in the known IRD genes, which are not easily identified by the targeted next-generation sequencing (NGS) or Sanger sequencing approaches. We hypothesized that copy-number variations (CNVs) are a major contributor to the elusive genetic causality of IRDs. METHODS: Twenty-eight cases previously unsolved with a targeted NGS were investigated with whole-genome single-nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) arrays. RESULTS: Deletions in the IRD genes were detected in 5 of 28 families, including a de novo deletion. We suggest that the de novo deletion occurred through nonallelic homologous recombination (NAHR) and we constructed a genomic map of NAHR-prone regions with overlapping IRD genes. In this article, we also report an unusual case of recessive retinitis pigmentosa due to compound heterozygous mutations in SNRNP200, a gene that is typically associated with the dominant form of this disease. CONCLUSIONS: CNV mapping substantially increased the genetic diagnostic rate of IRDs, detecting genetic causality in 18% of previously unsolved cases. Extending the search to other structural variations will probably demonstrate an even higher contribution to genetic causality of IRDs.Genet Med advance online publication 13 October 2016.

Disrupted lymphocyte homeostasis in hepatitis-associated acquired aplastic anemia is associated with short telomeres.

Hepatitis-associated aplastic anemia (HAA) is a variant of acquired aplastic anemia (AA) in which immune-mediated bone marrow failure (BMF) develops following an acute episode of seronegative hepatitis. Dyskeratosis congenita (DC) is an inherited BMF syndrome characterized by the presence of short telomeres, mucocutaneous abnormalities, and cancer predisposition. While both conditions may cause BMF and hepatic impairment, therapeutic approaches are distinct, making it imperative to establish the correct diagnosis. In clinical practice, lymphocyte telomere lengths (TL) are used as a first-line screen to rule out inherited telomeropathies before initiating treatment for AA. To evaluate the reliability of TL in the HAA population, we performed a retrospective analysis of TL in 10 consecutively enrolled HAA patients compared to 19 patients with idiopathic AA (IAA). HAA patients had significantly shorter telomeres than IAA patients (P = 0.009), including four patients with TL at or below the 1st percentile for age-matched controls. HAA patients had no clinical features of DC and did not carry disease-causing mutations in known genes associated with inherited telomere disorders. Instead, short TLs were significantly correlated with severe lymphopenia and skewed lymphocyte subsets, features characteristic of HAA. Our results indicate the importance of caution in the interpretation of TL measurements in HAA, because, in this patient population, short telomeres have limited specificity.

Single nucleotide polymorphism array analysis of bone marrow failure patients reveals characteristic patterns of genetic changes.

The bone marrow failure syndromes (BMFS) are a heterogeneous group of rare blood disorders characterized by inadequate haematopoiesis, clonal evolution, and increased risk of leukaemia. Single nucleotide polymorphism arrays (SNP-A) have been proposed as a tool for surveillance of clonal evolution in BMFS. To better understand the natural history of BMFS and to assess the clinical utility of SNP-A in these disorders, we analysed 124 SNP-A from a comprehensively characterized cohort of 91 patients at our BMFS centre. SNP-A were correlated with medical histories, haematopathology, cytogenetic and molecular data. To assess clonal evolution, longitudinal analysis of SNP-A was performed in 25 patients. We found that acquired copy number-neutral loss of heterozygosity (CN-LOH) was significantly more frequent in acquired aplastic anaemia (aAA) than in other BMFS (odds ratio 12·2, P < 0·01). Homozygosity by descent was most common in congenital BMFS, frequently unmasking autosomal recessive mutations. Copy number variants (CNVs) were frequently polymorphic, and we identified CNVs enriched in neutropenia and aAA. Our results suggest that acquired CN-LOH is a general phenomenon in aAA that is probably mechanistically and prognostically distinct from typical CN-LOH of myeloid malignancies. Our analysis of clinical utility of SNP-A shows the highest yield of detecting new clonal haematopoiesis at diagnosis and at relapse.

Analysis of chromosomal structural variation in patients with congenital left-sided cardiac lesions.

BACKGROUND: We sought to characterize the landscape of structural variation associated with the subset of congenital cardiac defects characterized by left-sided obstruction. METHODS: Cases with left-sided cardiac defects (LSCD) and pediatric controls were uniformly genotyped and assessed for copy number variant (CNV) calls. Significance testing was performed to ascertain differences in overall CNV incidence, and for CNV enrichment of specific genes and gene functions in LSCD cases relative to controls. RESULTS: A total of 257 cases of European descent and 962 ethnically matched, disease-free pediatric controls were included. Although there was no difference in CNV rate between cases and controls, a significant enrichment in rare LSCD CNVs was detected overall (p=7.30 × 10(-3) , case/control ratio=1.26) and when restricted either to deletions (p=7.58 × 10(-3) , case/control ratio=1.20) or duplications (3.02 × 10(-3) , case/control ratio=1.43). Neither gene-based, functional nor knowledge-based analyses identified genes, loci or pathways that were significantly enriched in cases as compared to controls when appropriate corrections for multiple tests were applied. However, several genes of interest were identified by virtue of their association with cardiac development, known human conditions, or reported disruption by CNVs in other patient cohorts. CONCLUSION: This study examines the largest cohort to date with LSCD for structural variation. These data suggest that CNVs play a role in disease risk and identify numerous genes disrupted by CNVs of potential disease relevance. These findings further highlight the genetic heterogeneity and complexity of these disorders.

Prevalence of rare mitochondrial DNA mutations in mitochondrial disorders.

BACKGROUND: Mitochondrial DNA (mtDNA) diseases are rare disorders whose prevalence is estimated around 1 in 5000. Patients are usually tested only for deletions and for common mutations of mtDNA which account for 5-40% of cases, depending on the study. However, the prevalence of rare mtDNA mutations is not known. METHODS: We analysed the whole mtDNA in a cohort of 743 patients suspected of manifesting a mitochondrial disease, after excluding deletions and common mutations. Both heteroplasmic and homoplasmic variants were identified using two complementary strategies (Surveyor and MitoChip). Multiple correspondence analyses followed by hierarchical ascendant cluster process were used to explore relationships between clinical spectrum, age at onset and localisation of mutations. RESULTS: 7.4% of deleterious mutations and 22.4% of novel putative mutations were identified. Pathogenic heteroplasmic mutations were more frequent than homoplasmic mutations (4.6% vs 2.8%). Patients carrying deleterious mutations showed symptoms before 16 years of age in 67% of cases. Early onset disease (<1 year) was significantly associated with mutations in protein coding genes (mainly in complex I) while late onset disorders (>16 years) were associated with mutations in tRNA genes. MTND5 and MTND6 genes were identified as 'hotspots' of mutations, with Leigh syndrome accounting for the large majority of associated phenotypes. CONCLUSIONS: Rare mitochondrial DNA mutations probably account for more than 7.4% of patients with respiratory chain deficiency. This study shows that a comprehensive analysis of mtDNA is essential, and should include young children, for an accurate diagnosis that is now accessible with the development of next generation sequencing technology.

FLT3 tyrosine kinase inhibition modulates PRC2 and promotes differentiation in acute myeloid leukemia.

Internal tandem duplication mutations in fms-like tyrosine kinase 3 (FLT3-ITD) are recurrent in acute myeloid leukemia (AML) and increase the risk of relapse. Clinical responses to FLT3 inhibitors (FLT3i) include myeloid differentiation of the FLT3-ITD clone in nearly half of patients through an unknown mechanism. We identified enhancer of zeste homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2), as a mediator of this effect using a proteomic-based screen. FLT3i downregulated EZH2 protein expression and PRC2 activity on H3K27me3. FLT3-ITD and loss-of-function mutations in EZH2 are mutually exclusive in human AML. We demonstrated that FLT3i increase myeloid maturation with reduced stem/progenitor cell populations in murine Flt3-ITD AML. Combining EZH1/2 inhibitors with FLT3i increased terminal maturation of leukemic cells and reduced leukemic burden. Our data suggest that reduced EZH2 activity following FLT3 inhibition promotes myeloid differentiation of FLT3-ITD leukemic cells, providing a mechanistic explanation for the clinical observations. These results demonstrate that in addition to its known cell survival and proliferation signaling, FLT3-ITD has a second, previously undefined function to maintain a myeloid stem/progenitor cell state through modulation of PRC2 activity. Our findings support exploring EZH1/2 inhibitors as therapy for FLT3-ITD AML.

Efficient digest of high-throughput sequencing data in a reproducible report.

BACKGROUND: High-throughput sequencing (HTS) technologies are spearheading the accelerated development of biomedical research. Processing and summarizing the large amount of data generated by HTS presents a non-trivial challenge to bioinformatics. A commonly adopted standard is to store sequencing reads aligned to a reference genome in SAM (Sequence Alignment/Map) or BAM (Binary Alignment/Map) files. Quality control of SAM/BAM files is a critical checkpoint before downstream analysis. The goal of the current project is to facilitate and standardize this process. RESULTS: We developed bamchop, a robust program to efficiently summarize key statistical metrics of HTS data stored in BAM files, and to visually present the results in a formatted report. The report documents information about various aspects of HTS data, such as sequencing quality, mapping to a reference genome, sequencing coverage, and base frequency. Bamchop uses the R language and Bioconductor packages to calculate statistical matrices and the Sweave utility and associated LaTeX markup for documentation. Bamchop's efficiency and robustness were tested on BAM files generated by local sequencing facilities and the 1000 Genomes Project. Source code, instruction and example reports of bamchop are freely available from https://github.com/CBMi-BiG/bamchop. CONCLUSIONS: Bamchop enables biomedical researchers to quickly and rigorously evaluate HTS data by providing a convenient synopsis and user-friendly reports.

Patient-derived Colonoids From Disease-spared Tissue Retain Inflammatory Bowel Disease-specific Transcriptomic Signatures.

BACKGROUND AND AIMS: A key histopathological feature of inflammatory bowel disease is damage to the mucosa, including breakdown of the epithelial barrier. Human enteroids and colonoids are a critical bench-to-bedside tool for studying the epithelium in inflammatory bowel disease. The goal of the current study was to define transcriptional differences in healthy versus diseased subjects that are sustained in enteroids and colonoids, including from disease-spared tissue. METHODS: Biopsies and matching enteroid or colonoid cultures from pediatric patients with ileal Crohn disease (N = 6) and control subjects (N = 17) were subjected to RNA sequencing followed by bioinformatic and machine learning analyses. Late passage enteroids were exposed to cytokines to assess durable transcriptional differences. RESULTS: We observed substantial overlap of pathways upregulated in Crohn disease in enteroids and ileal biopsies, as well as colonoids and rectal biopsies. KEGG pathways for cytokine-cytokine receptor interaction, chemokine signaling, protein export, and Toll-like receptor signaling were upregulated in both ileal and rectal biopsies, as well as enteroids and colonoids. In vitro cytokine exposure reactivated genes previously increased in biopsies. Machine learning predicted biopsy location (100% accuracy) and donor disease status (83% accuracy). A random forest classifier generated using ileal enteroids identified rectal colonoids from ileal Crohn disease subjects with 80% accuracy. CONCLUSION: We confirmed transcriptional profiles of Crohn disease biopsies are expressed in enteroids and colonoids. Furthermore, transcriptomic data from disease-spared rectal tissue can identify patients with ileal Crohn disease. Our data support the use of patient enteroids and colonoids as critical translational tools for the study of inflammatory bowel disease.

OpenPBTA: The Open Pediatric Brain Tumor Atlas.

Pediatric brain and spinal cancers are collectively the leading disease-related cause of death in children; thus, we urgently need curative therapeutic strategies for these tumors. To accelerate such discoveries, the Children's Brain Tumor Network (CBTN) and Pacific Pediatric Neuro-Oncology Consortium (PNOC) created a systematic process for tumor biobanking, model generation, and sequencing with immediate access to harmonized data. We leverage these data to establish OpenPBTA, an open collaborative project with over 40 scalable analysis modules that genomically characterize 1,074 pediatric brain tumors. Transcriptomic classification reveals universal TP53 dysregulation in mismatch repair-deficient hypermutant high-grade gliomas and TP53 loss as a significant marker for poor overall survival in ependymomas and H3 K28-mutant diffuse midline gliomas. Already being actively applied to other pediatric cancers and PNOC molecular tumor board decision-making, OpenPBTA is an invaluable resource to the pediatric oncology community.

HOXA Amplification Defines a Genetically Distinct Subset of Angiosarcomas.

Angiosarcoma is a rare, devastating malignancy with few curative options for disseminated disease. We analyzed a recently published genomic data set of 48 angiosarcomas and noticed recurrent amplifications of HOXA-cluster genes in 33% of patients. HOXA genes are master regulators of embryonic vascular development and adult neovascularization, which provides a molecular rationale to suspect that amplified HOXA genes act as oncogenes in angiosarcoma. HOXA amplifications typically affected multiple pro-angiogenic HOXA genes and co-occurred with amplifications of CD36 and KDR, whereas the overall mutation rate in these tumors was relatively low. HOXA amplifications were found most commonly in angiosarcomas located in the breast and were rare in angiosarcomas arising in sun-exposed areas on the head, neck, face and scalp. Our data suggest that HOXA-amplified angiosarcoma is a distinct molecular subgroup. Efforts to develop therapies targeting oncogenic HOX gene expression in AML and other sarcomas may have relevance for HOXA-amplified angiosarcoma.

Inducible Sbds deletion impairs bone marrow niche capacity to engraft donor bone marrow after transplantation.

Bone marrow (BM) niche-derived signals are critical for facilitating engraftment after hematopoietic stem cell (HSC) transplantation (HSCT). HSCT is required for restoration of hematopoiesis in patients with inherited BM failure syndromes (iBMFSs). Shwachman-Diamond syndrome (SDS) is a rare iBMFS associated with mutations in SBDS. Previous studies have demonstrated that SBDS deficiency in osteolineage niche cells causes BM dysfunction that promotes leukemia development. However, it is unknown whether BM niche defects caused by SBDS deficiency also impair efficient engraftment of healthy donor HSC after HSCT, a hypothesis that could explain morbidity noted after clinical HSCT for patients with SDS. Here, we report a mouse model with inducible Sbds deletion in hematopoietic and osteolineage cells. Primary and secondary BM transplantation (BMT) studies demonstrated that SBDS deficiency within BM niches caused poor donor hematopoietic recovery and specifically poor HSC engraftment after myeloablative BMT. We have also identified multiple molecular and cellular defects within niche populations that are driven by SBDS deficiency and are accentuated by or develop specifically after myeloablative conditioning. These abnormalities include altered frequencies of multiple niche cell subsets, including mesenchymal lineage cells, macrophages, and endothelial cells; disruption of growth factor signaling, chemokine pathway activation, and adhesion molecule expression; and p53 pathway activation and signals involved in cell cycle arrest. Taken together, this study demonstrates that SBDS deficiency profoundly impacts recipient hematopoietic niche function in the setting of HSCT, suggesting that novel therapeutic strategies targeting host niches could improve clinical HSCT outcomes for patients with SDS.

Pathogenicity and impact of HLA class I alleles in aplastic anemia patients of different ethnicities.

Acquired aplastic anemia (AA) is caused by autoreactive T cell-mediated destruction of early hematopoietic cells. Somatic loss of human leukocyte antigen (HLA) class I alleles was identified as a mechanism of immune escape in surviving hematopoietic cells of some patients with AA. However, pathogenicity, structural characteristics, and clinical impact of specific HLA alleles in AA remain poorly understood. Here, we evaluated somatic HLA loss in 505 patients with AA from 2 multi-institutional cohorts. Using a combination of HLA mutation frequencies, peptide-binding structures, and association with AA in an independent cohort of 6,323 patients from the National Marrow Donor Program, we identified 19 AA risk alleles and 12 non-risk alleles and established a potentially novel AA HLA pathogenicity stratification. Our results define pathogenicity for the majority of common HLA-A/B alleles across diverse populations. Our study demonstrates that HLA alleles confer different risks of developing AA, but once AA develops, specific alleles are not associated with response to immunosuppression or transplant outcomes. However, higher pathogenicity alleles, particularly HLA-B*14:02, are associated with higher rates of clonal evolution in adult patients with AA. Our study provides insights into the immune pathogenesis of AA, opening the door to future autoantigen identification and improved understanding of clonal evolution in AA.

Mitochondrial genome sequence analysis: a custom bioinformatics pipeline substantially improves Affymetrix MitoChip v2.0 call rate and accuracy.

BACKGROUND: Mitochondrial genome sequence analysis is critical to the diagnostic evaluation of mitochondrial disease. Existing methodologies differ widely in throughput, complexity, cost efficiency, and sensitivity of heteroplasmy detection. Affymetrix MitoChip v2.0, which uses a sequencing-by-genotyping technology, allows potentially accurate and high-throughput sequencing of the entire human mitochondrial genome to be completed in a cost-effective fashion. However, the relatively low call rate achieved using existing software tools has limited the wide adoption of this platform for either clinical or research applications. Here, we report the design and development of a custom bioinformatics software pipeline that achieves a much improved call rate and accuracy for the Affymetrix MitoChip v2.0 platform. We used this custom pipeline to analyze MitoChip v2.0 data from 24 DNA samples representing a broad range of tissue types (18 whole blood, 3 skeletal muscle, 3 cell lines), mutations (a 5.8 kilobase pair deletion and 6 known heteroplasmic mutations), and haplogroup origins. All results were compared to those obtained by at least one other mitochondrial DNA sequence analysis method, including Sanger sequencing, denaturing HPLC-based heteroduplex analysis, and/or the Illumina Genome Analyzer II next generation sequencing platform. RESULTS: An average call rate of 99.75% was achieved across all samples with our custom pipeline. Comparison of calls for 15 samples characterized previously by Sanger sequencing revealed a total of 29 discordant calls, which translates to an estimated 0.012% for the base call error rate. We successfully identified 4 known heteroplasmic mutations and 24 other potential heteroplasmic mutations across 20 samples that passed quality control. CONCLUSIONS: Affymetrix MitoChip v2.0 analysis using our optimized MitoChip Filtering Protocol (MFP) bioinformatics pipeline now offers the high sensitivity and accuracy needed for reliable, high-throughput and cost-efficient whole mitochondrial genome sequencing. This approach provides a viable alternative of potential utility for both clinical diagnostic and research applications to traditional Sanger and other emerging sequencing technologies for whole mitochondrial genome analysis.

Genome-Wide Association Studies of Conotruncal Heart Defects with Normally Related Great Vessels in the United States.

Conotruncal defects with normally related great vessels (CTD-NRGVs) occur in both patients with and without 22q11.2 deletion syndrome (22q11.2DS), but it is unclear to what extent the genetically complex etiologies of these heart defects may overlap across these two groups, potentially involving variation within and/or outside of the 22q11.2 region. To explore this potential overlap, we conducted genome-wide SNP-level, gene-level, and gene set analyses using common variants, separately in each of five cohorts, including two with 22q11.2DS (N = 1472 total cases) and three without 22q11.2DS (N = 935 total cases). Results from the SNP-level analyses were combined in meta-analyses, and summary statistics from these analyses were also used in gene and gene set analyses. Across all these analyses, no association was significant after correction for multiple comparisons. However, several SNPs, genes, and gene sets with suggestive evidence of association were identified. For common inherited variants, we did not identify strong evidence for shared genomic mechanisms for CTD-NRGVs across individuals with and without 22q11.2 deletions. Nevertheless, several of our top gene-level and gene set results have been linked to cardiogenesis and may represent candidates for future work.

Menin is necessary for long term maintenance of meningioma-1 driven leukemia.

Translocations of Meningioma-1 (MN1) occur in a subset of acute myeloid leukemias (AML) and result in high expression of MN1, either as a full-length protein, or as a fusion protein that includes most of the N-terminus of MN1. High levels of MN1 correlate with poor prognosis. When overexpressed in murine hematopoietic progenitors, MN1 causes an aggressive AML characterized by an aberrant myeloid precursor-like gene expression program that shares features of KMT2A-rearranged (KMT2A-r) leukemia, including high levels of Hoxa and Meis1 gene expression. Compounds that target a critical KMT2A-Menin interaction have proven effective in KMT2A-r leukemia. Here, we demonstrate that Menin (Men1) is also critical for the self-renewal of MN1-driven AML through the maintenance of a distinct gene expression program. Genetic inactivation of Men1 led to a decrease in the number of functional leukemia-initiating cells. Pharmacologic inhibition of the KMT2A-Menin interaction decreased colony-forming activity, induced differentiation programs in MN1-driven murine leukemia and decreased leukemic burden in a human AML xenograft carrying an MN1-ETV6 translocation. Collectively, these results nominate Menin inhibition as a promising therapeutic strategy in MN1-driven leukemia.

CNV Workshop: an integrated platform for high-throughput copy number variation discovery and clinical diagnostics.

BACKGROUND: Recent studies have shown that copy number variations (CNVs) are frequent in higher eukaryotes and associated with a substantial portion of inherited and acquired risk for various human diseases. The increasing availability of high-resolution genome surveillance platforms provides opportunity for rapidly assessing research and clinical samples for CNV content, as well as for determining the potential pathogenicity of identified variants. However, few informatics tools for accurate and efficient CNV detection and assessment currently exist. RESULTS: We developed a suite of software tools and resources (CNV Workshop) for automated, genome-wide CNV detection from a variety of SNP array platforms. CNV Workshop includes three major components: detection, annotation, and presentation of structural variants from genome array data. CNV detection utilizes a robust and genotype-specific extension of the Circular Binary Segmentation algorithm, and the use of additional detection algorithms is supported. Predicted CNVs are captured in a MySQL database that supports cohort-based projects and incorporates a secure user authentication layer and user/admin roles. To assist with determination of pathogenicity, detected CNVs are also annotated automatically for gene content, known disease loci, and gene-based literature references. Results are easily queried, sorted, filtered, and visualized via a web-based presentation layer that includes a GBrowse-based graphical representation of CNV content and relevant public data, integration with the UCSC Genome Browser, and tabular displays of genomic attributes for each CNV. CONCLUSIONS: To our knowledge, CNV Workshop represents the first cohesive and convenient platform for detection, annotation, and assessment of the biological and clinical significance of structural variants. CNV Workshop has been successfully utilized for assessment of genomic variation in healthy individuals and disease cohorts and is an ideal platform for coordinating multiple associated projects. AVAILABILITY AND IMPLEMENTATION: Available on the web at: http://sourceforge.net/projects/cnv.

Specific patterns of H3K79 methylation influence genetic interaction of oncogenes in AML.

Understanding mechanisms of cooperation between oncogenes is critical for the development of novel therapies and rational combinations. Acute myeloid leukemia (AML) cells with KMT2A-fusions and KMT2A partial tandem duplications (KMT2APTD) are known to depend on the histone methyltransferase DOT1L, which methylates histone 3 lysine 79 (H3K79). About 30% of KMT2APTD AMLs carry mutations in IDH1/2 (mIDH1/2). Previous studies showed that 2-hydroxyglutarate produced by mIDH1/2 increases H3K79 methylation, and mIDH1/2 patient samples are sensitive to DOT1L inhibition. Together, these findings suggested that stabilization or increases in H3K79 methylation associated with IDH mutations support the proliferation of leukemias dependent on this mark. However, we found that mIDH1/2 and KMT2A alterations failed to cooperate in an experimental model. Instead, mIDH1/2 and 2-hydroxyglutarate exert toxic effects, specifically on KMT2A-rearranged AML cells (fusions/partial tandem duplications). Mechanistically, we uncover an epigenetic barrier to efficient cooperation; mIDH1/2 expression is associated with high global histone 3 lysine 79 dimethylation (H3K79me2) levels, whereas global H3K79me2 is obligate low in KMT2A-rearranged AML. Increasing H3K79me2 levels, specifically in KMT2A-rearrangement leukemias, resulted in transcriptional downregulation of KMT2A target genes and impaired leukemia cell growth. Our study details a complex genetic and epigenetic interaction of 2 classes of oncogenes, IDH1/2 mutations and KMT2A rearrangements, that is unexpected based on the high percentage of IDH mutations in KMT2APTD AML. KMT2A rearrangements are associated with a trend toward lower response rates to mIDH1/2 inhibitors. The substantial adaptation that has to occur for 2 initially counteracting mutations to be tolerated within the same leukemic cell may provide at least a partial explanation for this observation.