Abyzov, Alexej

Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single-nucleotide polymorphism rs55705857, which confers a sixfold greater risk of isocitrate dehydrogenase ( IDH) –mutant low-grade glioma (LGG). We reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. Mutating the orthologous mouse rs55705857 locus accelerated tumor development in an Idh1 R132H -driven LGG mouse model from 472 to 172 days and increased penetrance from 30% to 75%. Our work reveals mechanisms of the heritable predisposition to lethal glioma in ~40% of LGG patients.

We analyzed 131 human brains (44 neurotypical, 19 with Tourette syndrome, 9 with schizophrenia, and 59 with autism) for somatic mutations after whole genome sequencing to a depth of more than 200×. Typically, brains had 20 to 60 detectable single-nucleotide mutations, but ~6% of brains harbored hundreds of somatic mutations. Hypermutability was associated with age and damaging mutations in genes implicated in cancers and, in some brains, reflected in vivo clonal expansions. Somatic duplications, likely arising during development, were found in ~5% of normal and diseased brains, reflecting background mutagenesis. Brains with autism were associated with mutations creating putative transcription factor binding motifs in enhancer-like regions in the developing brain. The top-ranked affected motifs corresponded to MEIS (myeloid ectopic viral integration site) transcription factors, suggesting a potential link between their involvement in gene regulation and autism.

Abstract Background Detecting copy number variations (CNVs) and copy number alterations (CNAs) based on whole-genome sequencing data is important for personalized genomics and treatment. CNVnator is one of the most popular tools for CNV/CNA discovery and analysis based on read depth. Findings Herein, we present an extension of CNVnator developed in Python—CNVpytor. CNVpytor inherits the reimplemented core engine of its predecessor and extends visualization, modularization, performance, and functionality. Additionally, CNVpytor uses B-allele frequency likelihood information from single-nucleotide polymorphisms and small indels data as additional evidence for CNVs/CNAs and as primary information for copy number–neutral losses of heterozygosity. Conclusions CNVpytor is significantly faster than CNVnator—particularly for parsing alignment files (2–20 times faster)—and has (20–50 times) smaller intermediate files. CNV calls can be filtered using several criteria, annotated, and merged over multiple samples. Modular architecture allows it to be used in shared and cloud environments such as Google Colab and Jupyter notebook. Data can be exported into JBrowse, while a lightweight plugin version of CNVpytor for JBrowse enables nearly instant and GUI-assisted analysis of CNVs by any user. CNVpytor release and the source code are available on GitHub at https://github.com/abyzovlab/CNVpytor under the MIT license.

Abstract BACKGROUND Determination of the causation of germline single nucleotide polymorphisms (SNPs) located in non-coding regions of the genome is challenging. The genomic region of 8q24 has been identified as important in many kinds of cancer, linked to a topologically associated domain (TAD) encompassing MYC; this TAD contains a GWAS SNP (rs55705857) associated with IDH-mutant glioma. METHODS Germline genotyping data from 622 IDH-mutant glioma and 668 controls were used to fine map the rs55705857 locus by detailed haplotype analysis. Chromatin immunoprecipitation sequencing (ChIP-seq) of histone markers H3K4me1, H3K4me3, H3K27ac and H3K36me3 was performed on normal brain samples (n=8) and human glioma samples (n=11 IDH-wt and 52 IDH-mut). RNAseq from 9 normal and 83 brain tumors (n=26 IDH-wt and 55 IDH-mut) were used to assess differential gene expression. RESULTS Fine-mapping identified rs55705857 SNP as the most likely causative allele (OR=8.69; p<0.001) within 8q24 for the development of IDH-mutant glioma. At rs55705857, both H3K27ac and H3K4me1 in IDH-mutant vs IDH-wt tumors were increased 3.05- and 1.58-fold, respectively (DiffBind; p=5.81×10-7 and p=2.31×10-3). ChromHMM analysis of the marks indicated that promoter and enhancer functions were significantly increased, and the activity broadened at rs55705857 in IDH-mut gliomas compared to IDH-wt tumors and normal brain samples. This enhancement correlated with significant increased MYC expression in IDH-mut gliomas (p=3.1×10-13), as well as alterations of Myc signaling targets. Publicly available ATACseq, ChIPseq and long-range DNA interaction data demonstrated that the rs55705857 locus is open and interacts with the MYC promoter. CONCLUSIONS Fine-mapping of the 8q24 locus provided strong evidence that rs55705857 is the causative 8q24 locus associated with IDH-mut glioma. Functional experiments suggest that IDH mutation facilitates rs55705857 interaction with MYC to alter downstream MYC targets.

Abstract Establishing causal links between genetic polymorphisms and increased heritable risk of developing brain cancer is a major challenge. The non-coding single nucleotide polymorphism rs55705857 (A >G) is associated with a ~6-fold increased risk to develop IDH-mutant low-grade glioma (LGG). The rs55705857 G allele has a minor allele frequency of only ~5% in the general population but is found in ~40% of patients with IDH-mutant LGG and patients carrying risk-alleles are diagnosed on average 7-12 years earlier than those carrying non-risk A alleles. This makes rs55705857 one of the highest reported genetic associations with cancer, comparable with inherited BRCA1 gene mutations and the risk of developing breast cancer or other familial glioma genes such as NF1/2, CDKN2A or p53. To generate a LGG mouse model, we combined clonal activation of IDH1R132H with mutations of Trp53 and Atrx, which resulted in the development of LGG-like brain tumors in 25% of mice. Mutating the highly conserved, orthologous mouse rs55705857 locus to mimic the human risk allele dramatically accelerated tumor development from 463 to 172 days and increased penetrance to 75%. The resulting tumors exhibit elevated R-2-hydroxyglutarate levels, well-differentiated fibrillary neoplastic histology and metabolic rewiring, recapitulating histopathological and molecular hallmarks of human LGG. Mechanistically, we show that the rs55705857 locus resides within a brain-specific enhancer, which shows enhanced activity in IDH-mutant tumors. In addition, we found that the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. The hyperactive chromatin status combined with the tissue specificity of this enhancer explains the cooperativity between mutant IDH and rs55705857 and why rs55705857 is associated specifically with IDH-mutant glioma, but not other cancers. Overall, we generated new LGG mouse models, which provide insights into the pathophysiology of this deadly disease and shed light into the heritable predisposition to LGG development.

Mutations provide an enduring record Somatic mutations pepper our cells with change, but because they are not in the germline, they do not propagate to the next generation. Bizzotto et al. leveraged data on the distribution of somatic mutations in adults to take a backward look at the earliest moments of human development. Calculation of cellular lineages on the basis of shared somatic mutations shows the number of cells from which the body will develop when the human embryo gastrulates. The lineage for forebrain cells is identifiable, as are the asymmetrical fates spun out of many of the gastrula cells. Science , this issue p. 1249

Genomic sleuthing spots early divergence After fertilization, the human zygote divides into two cells. Fasching et al. used genomic analysis from cellular samples taken much later in development to back-calculate the cell division trees that went before. Although the first cell division in human development looks symmetrical from the outside, the fates followed by daughter cells from each of those first two blastomeres are anything but the same. As much as 90% of blood cells are derived from just one of the first two blastomeres. Science , this issue p. 1245

Abstract Background: Genome-wide association studies (GWAS) revealed that 25 regions in 24 genes are associated with adult diffuse glioma development. These regions were identified by performing GWAS of glioma overall and by pathology (GBM and nonGBM). The 2016 WHO Classification of Tumors of the Central Nervous System utilizes two somatic alterations to molecularly-classify adult diffuse glioma: IDH mutation and 1p/19q codeletion. We hypothesized that germline variants may increase susceptibility to, or interact with, these somatic alterations to accelerate the development of specific molecular subtypes of glioma. We further hypothesize that germline variants associated with IDH-mutated glioma might be associated with other IDH-mutated tumors, namely, cholangiocarcinoma, acute myeloid leukemia (AML) and melanoma. Methods: We performed a GWAS by glioma molecular subtype - as defined by presence or absence of IDH somatic mutation and 1p/19q codeletion. A total of 1320 glioma cases and 1889 controls were used in the discovery set, and 799 glioma cases and 808 controls in the validation set. A meta-analysis was performed with a genome-wide p-value threshold of 5 × 10−8. GTEx data were used to perform an expression quantitative trait loci (eQTL) analysis. For germline variants that were significantly associated with IDH-mutated glioma, we evaluated pleiotropy with cholangiocarcinoma, AML and melanoma using TCGA and Mayo Biobank controls. Results: Variants in or near D2HGDH on chromosome 2 were genome-wide significant in IDH-mutated glioma (meta p-value = 2.82 × 10−10). TCGA reported that the D2HGDH region was commonly deleted in IDH-mutated gliomas that do not have 1p/19q codeletion. In TCGA data for IDH-mutated, non-codeleted glioma, we observed that the D2HGDH variant was inversely associated with tumor deletions of D2HGDH (odds ratio=0.57, p-value=0.015). The eQTL analyses demonstrated significant associations between D2HGDH germline variant and expression of D2HGDH (p=2.2 × 10−11). Further stratifying IDH-mutated glioma by 1p/19q codeletion status, one variant near FAM20C on chromosome 7 was genome-wide significant in gliomas that have IDH mutation and 1p/19q codeletion (meta p-value=9.56 × 10−9). Analyses are currently underway to evaluate pleiotropy of these IDH-mutated glioma germline variants with other IDH-mutated tumors including cholangiocarcinoma, AML and melanoma. Variants in or near GMEB2 on chromosome 20 were genome-wide significant in IDH wild-type glioma (meta p-value=2.60 × 10−10). The most significant variant in the GMEB2 region remained significant after adjustment for the known RTEL1 glioma risk variant nearby on chromosome 20 (p=0.029). Conclusions: We identified and validated novel germline variants in two genes that are associated with etiology of IDH-mutated and one gene that is associated with IDH wild-type adult diffuse glioma. Citation Format: Jeanette E. Eckel-Passow, Kristen L. Drucker, Thomas M. Kollmeyer, Matthew L. Kosel, Paul A. Decker, Annette M. Molinaro, Terri Rice, Corrine E. Praska, Lauren E. Clark, Alissa A. Caron, Alexej Abyzov, Anthony Batzler, Jun S. Song, Melike Pekmezci, Helen M. Hansen, Lucie S. McCoy, Paige M. Bracci, Joseph Wiemels, John K. Wiencke, Stephen Francis, Terence C. Burns, Caterina Giannini, Daniel H. Lachance, Margaret Wrensch, Robert B. Jenkins. Adult diffuse glioma GWAS by molecular subtype identifies variants in D2HGDH, FAM20C and GMEB2 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1193.

Abstract Summary Defining the precise location of structural variations (SVs) at single-nucleotide breakpoint resolution is a challenging problem due to large gaps in alignment. Previously, Alignment with Gap Excision (AGE) enabled us to define breakpoints of SVs at single-nucleotide resolution; however, AGE requires a vast amount of memory when aligning a pair of long sequences. To address this, we developed a memory-efficient implementation—LongAGE—based on the classical Hirschberg algorithm. We demonstrate an application of LongAGE for resolving breakpoints of SVs embedded into segmental duplications on Pacific Biosciences (PacBio) reads that can be longer than 10 kb. Furthermore, we observed different breakpoints for a deletion and a duplication in the same locus, providing direct evidence that such multi-allelic copy number variants (mCNVs) arise from two or more independent ancestral mutations. Availability and implementation LongAGE is implemented in C++ and available on Github at https://github.com/Coaxecva/LongAGE. Supplementary information Supplementary data are available at Bioinformatics online.