Loss of chromosome Y in primary tumors.

Certain cancer types afflict female and male patients disproportionately. The reasons include differences in male/female physiology, effect of sex hormones, risk behavior, environmental exposures, and genetics of the sex chromosomes X and Y. Loss of Y (LOY) is common in peripheral blood cells in aging men, and this phenomenon is associated with several diseases. However, the frequency and role of LOY in tumors is little understood. Here, we present a comprehensive catalog of LOY in >5,000 primary tumors from male patients in the TCGA. We show that LOY rates vary by tumor type and provide evidence for LOY being either a passenger or driver event depending on context. LOY in uveal melanoma specifically is associated with age and survival and is an independent predictor of poor outcome. LOY creates common dependencies on DDX3X and EIF1AX in male cell lines, suggesting that LOY generates unique vulnerabilities that could be therapeutically exploited.

Influences of rare copy-number variation on human complex traits.

The human genome contains hundreds of thousands of regions harboring copy-number variants (CNV). However, the phenotypic effects of most such polymorphisms are unknown because only larger CNVs have been ascertainable from SNP-array data generated by large biobanks. We developed a computational approach leveraging haplotype sharing in biobank cohorts to more sensitively detect CNVs. Applied to UK Biobank, this approach accounted for approximately half of all rare gene inactivation events produced by genomic structural variation. This CNV call set enabled a detailed analysis of associations between CNVs and 56 quantitative traits, identifying 269 independent associations (p < 5 × 10-8) likely to be causally driven by CNVs. Putative target genes were identifiable for nearly half of the loci, enabling insights into dosage sensitivity of these genes and uncovering several gene-trait relationships. These results demonstrate the ability of haplotype-informed analysis to provide insights into the genetic basis of human complex traits.

Genomic architecture of autism from comprehensive whole-genome sequence annotation.

Fully understanding autism spectrum disorder (ASD) genetics requires whole-genome sequencing (WGS). We present the latest release of the Autism Speaks MSSNG resource, which includes WGS data from 5,100 individuals with ASD and 6,212 non-ASD parents and siblings (total n = 11,312). Examining a wide variety of genetic variants in MSSNG and the Simons Simplex Collection (SSC; n = 9,205), we identified ASD-associated rare variants in 718/5,100 individuals with ASD from MSSNG (14.1%) and 350/2,419 from SSC (14.5%). Considering genomic architecture, 52% were nuclear sequence-level variants, 46% were nuclear structural variants (including copy-number variants, inversions, large insertions, uniparental isodisomies, and tandem repeat expansions), and 2% were mitochondrial variants. Our study provides a guidebook for exploring genotype-phenotype correlations in families who carry ASD-associated rare variants and serves as an entry point to the expanded studies required to dissect the etiology in the ∼85% of the ASD population that remain idiopathic.

A cross-disorder dosage sensitivity map of the human genome.

Rare copy-number variants (rCNVs) include deletions and duplications that occur infrequently in the global human population and can confer substantial risk for disease. In this study, we aimed to quantify the properties of haploinsufficiency (i.e., deletion intolerance) and triplosensitivity (i.e., duplication intolerance) throughout the human genome. We harmonized and meta-analyzed rCNVs from nearly one million individuals to construct a genome-wide catalog of dosage sensitivity across 54 disorders, which defined 163 dosage sensitive segments associated with at least one disorder. These segments were typically gene dense and often harbored dominant dosage sensitive driver genes, which we were able to prioritize using statistical fine-mapping. Finally, we designed an ensemble machine-learning model to predict probabilities of dosage sensitivity (pHaplo & pTriplo) for all autosomal genes, which identified 2,987 haploinsufficient and 1,559 triplosensitive genes, including 648 that were uniquely triplosensitive. This dosage sensitivity resource will provide broad utility for human disease research and clinical genetics.

Pan-genome analysis of 33 genetically diverse rice accessions reveals hidden genomic variations.

Structural variations (SVs) and gene copy number variations (gCNVs) have contributed to crop evolution, domestication, and improvement. Here, we assembled 31 high-quality genomes of genetically diverse rice accessions. Coupling with two existing assemblies, we developed pan-genome-scale genomic resources including a graph-based genome, providing access to rice genomic variations. Specifically, we discovered 171,072 SVs and 25,549 gCNVs and used an Oryza glaberrima assembly to infer the derived states of SVs in the Oryza sativa population. Our analyses of SV formation mechanisms, impacts on gene expression, and distributions among subpopulations illustrate the utility of these resources for understanding how SVs and gCNVs shaped rice environmental adaptation and domestication. Our graph-based genome enabled genome-wide association study (GWAS)-based identification of phenotype-associated genetic variations undetectable when using only SNPs and a single reference assembly. Our work provides rich population-scale resources paired with easy-to-access tools to facilitate rice breeding as well as plant functional genomics and evolutionary biology research.

De novo deletions and duplications at recombination hotspots in mouse germlines.

Numerous DNA double-strand breaks (DSBs) arise during meiosis to initiate homologous recombination. These DSBs are usually repaired faithfully, but here, we uncover a distinct type of mutational event in which deletions form via joining of ends from two closely spaced DSBs (double cuts) within a single hotspot or at adjacent hotspots on the same or different chromatids. Deletions occur in normal meiosis but are much more frequent when DSB formation is dysregulated in the absence of the ATM kinase. Events between chromosome homologs point to multi-chromatid damage and aborted gap repair. Some deletions contain DNA from other hotspots, indicating that double cutting at distant sites creates substrates for insertional mutagenesis. End joining at double cuts can also yield tandem duplications or extrachromosomal circles. Our findings highlight the importance of DSB regulation and reveal a previously hidden potential for meiotic mutagenesis that is likely to affect human health and genome evolution.

Major Impacts of Widespread Structural Variation on Gene Expression and Crop Improvement in Tomato.

Structural variants (SVs) underlie important crop improvement and domestication traits. However, resolving the extent, diversity, and quantitative impact of SVs has been challenging. We used long-read nanopore sequencing to capture 238,490 SVs in 100 diverse tomato lines. This panSV genome, along with 14 new reference assemblies, revealed large-scale intermixing of diverse genotypes, as well as thousands of SVs intersecting genes and cis-regulatory regions. Hundreds of SV-gene pairs exhibit subtle and significant expression changes, which could broadly influence quantitative trait variation. By combining quantitative genetics with genome editing, we show how multiple SVs that changed gene dosage and expression levels modified fruit flavor, size, and production. In the last example, higher order epistasis among four SVs affecting three related transcription factors allowed introduction of an important harvesting trait in modern tomato. Our findings highlight the underexplored role of SVs in genotype-to-phenotype relationships and their widespread importance and utility in crop improvement.

Converting Escherichia coli to a Synthetic Methylotroph Growing Solely on Methanol.

Methanol, being electron rich and derivable from methane or CO2, is a potentially renewable one-carbon (C1) feedstock for microorganisms. Although the ribulose monophosphate (RuMP) cycle used by methylotrophs to assimilate methanol differs from the typical sugar metabolism by only three enzymes, turning a non-methylotrophic organism to a synthetic methylotroph that grows to a high cell density has been challenging. Here we reprogrammed E. coli using metabolic robustness criteria followed by laboratory evolution to establish a strain that can efficiently utilize methanol as the sole carbon source. This synthetic methylotroph alleviated a so far uncharacterized hurdle, DNA-protein crosslinking (DPC), by insertion sequence (IS)-mediated copy number variations (CNVs) and balanced the metabolic flux by mutations. Being capable of growing at a rate comparable with natural methylotrophs in a wide range of methanol concentrations, this synthetic methylotrophic strain illustrates genome editing and evolution for microbial tropism changes and expands the scope of biological C1 conversion.

Clonal Decomposition and DNA Replication States Defined by Scaled Single-Cell Genome Sequencing.

Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.

A Mild PUM1 Mutation Is Associated with Adult-Onset Ataxia, whereas Haploinsufficiency Causes Developmental Delay and Seizures.

Certain mutations can cause proteins to accumulate in neurons, leading to neurodegeneration. We recently showed, however, that upregulation of a wild-type protein, Ataxin1, caused by haploinsufficiency of its repressor, the RNA-binding protein Pumilio1 (PUM1), also causes neurodegeneration in mice. We therefore searched for human patients with PUM1 mutations. We identified eleven individuals with either PUM1 deletions or de novo missense variants who suffer a developmental syndrome (Pumilio1-associated developmental disability, ataxia, and seizure; PADDAS). We also identified a milder missense mutation in a family with adult-onset ataxia with incomplete penetrance (Pumilio1-related cerebellar ataxia, PRCA). Studies in patient-derived cells revealed that the missense mutations reduced PUM1 protein levels by ∼25% in the adult-onset cases and by ∼50% in the infantile-onset cases; levels of known PUM1 targets increased accordingly. Changes in protein levels thus track with phenotypic severity, and identifying posttranscriptional modulators of protein expression should identify new candidate disease genes.

Multiclonal Invasion in Breast Tumors Identified by Topographic Single Cell Sequencing.

Ductal carcinoma in situ (DCIS) is an early-stage breast cancer that infrequently progresses to invasive ductal carcinoma (IDC). Genomic evolution has been difficult to delineate during invasion due to intratumor heterogeneity and the low number of tumor cells in the ducts. To overcome these challenges, we developed Topographic Single Cell Sequencing (TSCS) to measure genomic copy number profiles of single tumor cells while preserving their spatial context in tissue sections. We applied TSCS to 1,293 single cells from 10 synchronous patients with both DCIS and IDC regions in addition to exome sequencing. Our data reveal a direct genomic lineage between in situ and invasive tumor subpopulations and further show that most mutations and copy number aberrations evolved within the ducts prior to invasion. These results support a multiclonal invasion model, in which one or more clones escape the ducts and migrate into the adjacent tissues to establish the invasive carcinomas.

Chemoresistance Evolution in Triple-Negative Breast Cancer Delineated by Single-Cell Sequencing.

Triple-negative breast cancer (TNBC) is an aggressive subtype that frequently develops resistance to chemotherapy. An unresolved question is whether resistance is caused by the selection of rare pre-existing clones or alternatively through the acquisition of new genomic aberrations. To investigate this question, we applied single-cell DNA and RNA sequencing in addition to bulk exome sequencing to profile longitudinal samples from 20 TNBC patients during neoadjuvant chemotherapy (NAC). Deep-exome sequencing identified 10 patients in which NAC led to clonal extinction and 10 patients in which clones persisted after treatment. In 8 patients, we performed a more detailed study using single-cell DNA sequencing to analyze 900 cells and single-cell RNA sequencing to analyze 6,862 cells. Our data showed that resistant genotypes were pre-existing and adaptively selected by NAC, while transcriptional profiles were acquired by reprogramming in response to chemotherapy in TNBC patients.

Immunoglobulin germline gene polymorphisms influence the function of SARS-CoV-2 neutralizing antibodies.

The human immunoglobulin heavy-chain (IGH) locus is exceptionally polymorphic, with high levels of allelic and structural variation. Thus, germline IGH genotypes are personal, which may influence responses to infection and vaccination. For an improved understanding of inter-individual differences in antibody responses, we isolated SARS-CoV-2 spike-specific monoclonal antibodies from convalescent health care workers, focusing on the IGHV1-69 gene, which has the highest level of allelic variation of all IGHV genes. The IGHV1-69∗20-using CAB-I47 antibody and two similar antibodies isolated from an independent donor were critically dependent on allele usage. Neutralization was retained when reverting the V region to the germline IGHV1-69∗20 allele but lost when reverting to other IGHV1-69 alleles. Structural data confirmed that two germline-encoded polymorphisms, R50 and F55, in the IGHV1-69 gene were required for high-affinity receptor-binding domain interaction. These results demonstrate that polymorphisms in IGH genes can influence the function of SARS-CoV-2 neutralizing antibodies.

Allele-Specific HLA Loss and Immune Escape in Lung Cancer Evolution.

Immune evasion is a hallmark of cancer. Losing the ability to present neoantigens through human leukocyte antigen (HLA) loss may facilitate immune evasion. However, the polymorphic nature of the locus has precluded accurate HLA copy-number analysis. Here, we present loss of heterozygosity in human leukocyte antigen (LOHHLA), a computational tool to determine HLA allele-specific copy number from sequencing data. Using LOHHLA, we find that HLA LOH occurs in 40% of non-small-cell lung cancers (NSCLCs) and is associated with a high subclonal neoantigen burden, APOBEC-mediated mutagenesis, upregulation of cytolytic activity, and PD-L1 positivity. The focal nature of HLA LOH alterations, their subclonal frequencies, enrichment in metastatic sites, and occurrence as parallel events suggests that HLA LOH is an immune escape mechanism that is subject to strong microenvironmental selection pressures later in tumor evolution. Characterizing HLA LOH with LOHHLA refines neoantigen prediction and may have implications for our understanding of resistance mechanisms and immunotherapeutic approaches targeting neoantigens. VIDEO ABSTRACT.

Loss of IFN-γ Pathway Genes in Tumor Cells as a Mechanism of Resistance to Anti-CTLA-4 Therapy.

Antibody blockade of the inhibitory CTLA-4 pathway has led to clinical benefit in a subset of patients with metastatic melanoma. Anti-CTLA-4 enhances T cell responses, including production of IFN-γ, which is a critical cytokine for host immune responses. However, the role of IFN-γ signaling in tumor cells in the setting of anti-CTLA-4 therapy remains unknown. Here, we demonstrate that patients identified as non-responders to anti-CTLA-4 (ipilimumab) have tumors with genomic defects in IFN-γ pathway genes. Furthermore, mice bearing melanoma tumors with knockdown of IFN-γ receptor 1 (IFNGR1) have impaired tumor rejection upon anti-CTLA-4 therapy. These data highlight that loss of the IFN-γ signaling pathway is associated with primary resistance to anti-CTLA-4 therapy. Our findings demonstrate the importance of tumor genomic data, especially IFN-γ related genes, as prognostic information for patients selected to receive treatment with immune checkpoint therapy.

Kinetic Analysis of Protein Stability Reveals Age-Dependent Degradation.

Do young and old protein molecules have the same probability to be degraded? We addressed this question using metabolic pulse-chase labeling and quantitative mass spectrometry to obtain degradation profiles for thousands of proteins. We find that >10% of proteins are degraded non-exponentially. Specifically, proteins are less stable in the first few hours of their life and stabilize with age. Degradation profiles are conserved and similar in two cell types. Many non-exponentially degraded (NED) proteins are subunits of complexes that are produced in super-stoichiometric amounts relative to their exponentially degraded (ED) counterparts. Within complexes, NED proteins have larger interaction interfaces and assemble earlier than ED subunits. Amplifying genes encoding NED proteins increases their initial degradation. Consistently, decay profiles can predict protein level attenuation in aneuploid cells. Together, our data show that non-exponential degradation is common, conserved, and has important consequences for complex formation and regulation of protein abundance.

The Dynamic Fungal Genome: Polyploidy, Aneuploidy and Copy Number Variation in Response to Stress.

Fungal species have dynamic genomes and often exhibit genomic plasticity in response to stress. This genome plasticity often comes with phenotypic consequences that affect fitness and resistance to stress. Fungal pathogens exhibit genome plasticity in both clinical and agricultural settings and often during adaptation to antifungal drugs, posing significant challenges to human health. Therefore, it is important to understand the rates, mechanisms, and impact of large genomic changes. This review addresses the prevalence of polyploidy, aneuploidy, and copy number variation across diverse fungal species, with special attention to prominent fungal pathogens and model species. We also explore the relationship between environmental stress and rates of genomic changes and highlight the mechanisms underlying genotypic and phenotypic changes. A comprehensive understanding of these dynamic fungal genomes is needed to identify novel solutions for the increase in antifungal drug resistance.

A Highly Scalable Method for Joint Whole-Genome Sequencing and Gene-Expression Profiling of Single Cells.

To address how genetic variation alters gene expression in complex cell mixtures, we developed direct nuclear tagmentation and RNA sequencing (DNTR-seq), which enables whole-genome and mRNA sequencing jointly in single cells. DNTR-seq readily identified minor subclones within leukemia patients. In a large-scale DNA damage screen, DNTR-seq was used to detect regions under purifying selection and identified genes where mRNA abundance was resistant to copy-number alteration, suggesting strong genetic compensation. mRNA sequencing (mRNA-seq) quality equals RNA-only methods, and the low positional bias of genomic libraries allowed detection of sub-megabase aberrations at ultra-low coverage. Each cell library is individually addressable and can be re-sequenced at increased depth, allowing multi-tiered study designs. Additionally, the direct tagmentation protocol enables coverage-independent estimation of ploidy, which can be used to identify cell singlets. Thus, DNTR-seq directly links each cell's state to its corresponding genome at scale, enabling routine analysis of heterogeneous tumors and other complex tissues.

Exome copy number variant detection, analysis, and classification in a large cohort of families with undiagnosed rare genetic disease.

Copy number variants (CNVs) are significant contributors to the pathogenicity of rare genetic diseases and, with new innovative methods, can now reliably be identified from exome sequencing. Challenges still remain in accurate classification of CNV pathogenicity. CNV calling using GATK-gCNV was performed on exomes from a cohort of 6,633 families (15,759 individuals) with heterogeneous phenotypes and variable prior genetic testing collected at the Broad Institute Center for Mendelian Genomics of the Genomics Research to Elucidate the Genetics of Rare Diseases consortium and analyzed using the seqr platform. The addition of CNV detection to exome analysis identified causal CNVs for 171 families (2.6%). The estimated sizes of CNVs ranged from 293 bp to 80 Mb. The causal CNVs consisted of 140 deletions, 15 duplications, 3 suspected complex structural variants (SVs), 3 insertions, and 10 complex SVs, the latter two groups being identified by orthogonal confirmation methods. To classify CNV variant pathogenicity, we used the 2020 American College of Medical Genetics and Genomics/ClinGen CNV interpretation standards and developed additional criteria to evaluate allelic and functional data as well as variants on the X chromosome to further advance the framework. We interpreted 151 CNVs as likely pathogenic/pathogenic and 20 CNVs as high-interest variants of uncertain significance. Calling CNVs from existing exome data increases the diagnostic yield for individuals undiagnosed after standard testing approaches, providing a higher-resolution alternative to arrays at a fraction of the cost of genome sequencing. Our improvements to the classification approach advances the systematic framework to assess the pathogenicity of CNVs.

A comprehensive analysis of clinical and polygenic germline influences on somatic mutational burden.

Tumor mutational burden (TMB), the total number of somatic mutations in the tumor, and copy number burden (CNB), the corresponding measure of aneuploidy, are established fundamental somatic features and emerging biomarkers for immunotherapy. However, the genetic and non-genetic influences on TMB/CNB and, critically, the manner by which they influence patient outcomes remain poorly understood. Here, we present a large germline-somatic study of TMB/CNB with >23,000 individuals across 17 cancer types, of which 12,000 also have extensive clinical, treatment, and overall survival (OS) measurements available. We report dozens of clinical associations with TMB/CNB, observing older age and male sex to have a strong effect on TMB and weaker impact on CNB. We additionally identified significant germline influences on TMB/CNB, including fine-scale European ancestry and germline polygenic risk scores (PRSs) for smoking, tanning, white blood cell counts, and educational attainment. We quantify the causal effect of exposures on somatic mutational processes using Mendelian randomization. Many of the identified features associated with TMB/CNB were additionally associated with OS for individuals treated at a single tertiary cancer center. For individuals receiving immunotherapy, we observed a complex relationship between PRSs for educational attainment, self-reported college attainment, TMB, and survival, suggesting that the influence of this biomarker may be substantially modified by socioeconomic status. While the accumulation of somatic alterations is a stochastic process, our work demonstrates that it can be shaped by host characteristics including germline genetics.