ABSTRACT
Primary open-angle glaucoma (POAG), the leading cause of irreversible blindness worldwide, disproportionately affects individuals of African ancestry. We conducted a genome-wide association study (GWAS) for POAG in 11,275 individuals of African ancestry (6,003 cases; 5,272 controls). We detected 46 risk loci associated with POAG at genome-wide significance. Replication and post-GWAS analyses, including functionally informed fine-mapping, multiple trait co-localization, and in silico validation, implicated two previously undescribed variants (rs1666698 mapping to DBF4P2; rs34957764 mapping to ROCK1P1) and one previously associated variant (rs11824032 mapping to ARHGEF12) as likely causal. For individuals of African ancestry, a polygenic risk score (PRS) for POAG from our mega-analysis (African ancestry individuals) outperformed a PRS from summary statistics of a much larger GWAS derived from European ancestry individuals. This study quantifies the genetic architecture similarities and differences between African and non-African ancestry populations for this blinding disease.
Subject(s)
Genome-Wide Association Study , Glaucoma, Open-Angle , Humans , Genetic Predisposition to Disease , Glaucoma, Open-Angle/genetics , Black People/genetics , Polymorphism, Single Nucleotide/geneticsABSTRACT
Although the blinding eye disease glaucoma is more common in people of African ancestry, previous genetic studies predominantly involved European subjects. In this issue of Cell, O'Brien et al. report a genome-wide association study for glaucoma in individuals of African ancestry, showing overlap with European studies and refining an African polygenic risk score.
Subject(s)
Genome-Wide Association Study , Glaucoma , Humans , Glaucoma/genetics , Black People/genetics , Research , Genetic Predisposition to Disease , Polymorphism, Single NucleotideABSTRACT
Characterizing somatic mutations in the brain is important for disentangling the complex mechanisms of aging, yet little is known about mutational patterns in different brain cell types. Here, we performed whole-genome sequencing (WGS) of 86 single oligodendrocytes, 20 mixed glia, and 56 single neurons from neurotypical individuals spanning 0.4-104 years of age and identified >92,000 somatic single-nucleotide variants (sSNVs) and small insertions/deletions (indels). Although both cell types accumulate somatic mutations linearly with age, oligodendrocytes accumulated sSNVs 81% faster than neurons and indels 28% slower than neurons. Correlation of mutations with single-nucleus RNA profiles and chromatin accessibility from the same brains revealed that oligodendrocyte mutations are enriched in inactive genomic regions and are distributed across the genome similarly to mutations in brain cancers. In contrast, neuronal mutations are enriched in open, transcriptionally active chromatin. These stark differences suggest an assortment of active mutagenic processes in oligodendrocytes and neurons.
Subject(s)
Aging , Brain , Neurons , Oligodendroglia , Humans , Aging/genetics , Aging/pathology , Chromatin/genetics , Chromatin/metabolism , Mutation , Neurons/metabolism , Neurons/pathology , Oligodendroglia/metabolism , Oligodendroglia/pathology , Single-Cell Gene Expression Analysis , Whole Genome Sequencing , Brain/metabolism , Brain/pathology , Polymorphism, Single Nucleotide , INDEL Mutation , Biological Specimen Banks , Oligodendrocyte Precursor Cells/metabolism , Oligodendrocyte Precursor Cells/pathologyABSTRACT
Understanding how genetic variants impact molecular phenotypes is a key goal of functional genomics, currently hindered by reliance on a single haploid reference genome. Here, we present the EN-TEx resource of 1,635 open-access datasets from four donors (â¼30 tissues × â¼15 assays). The datasets are mapped to matched, diploid genomes with long-read phasing and structural variants, instantiating a catalog of >1 million allele-specific loci. These loci exhibit coordinated activity along haplotypes and are less conserved than corresponding, non-allele-specific ones. Surprisingly, a deep-learning transformer model can predict the allele-specific activity based only on local nucleotide-sequence context, highlighting the importance of transcription-factor-binding motifs particularly sensitive to variants. Furthermore, combining EN-TEx with existing genome annotations reveals strong associations between allele-specific and GWAS loci. It also enables models for transferring known eQTLs to difficult-to-profile tissues (e.g., from skin to heart). Overall, EN-TEx provides rich data and generalizable models for more accurate personal functional genomics.
Subject(s)
Epigenome , Quantitative Trait Loci , Genome-Wide Association Study , Genomics , Phenotype , Polymorphism, Single NucleotideABSTRACT
Autozygosity is associated with rare Mendelian disorders and clinically relevant quantitative traits. We investigated associations between the fraction of the genome in runs of homozygosity (FROH) and common diseases in Genes & Health (n = 23,978 British South Asians), UK Biobank (n = 397,184), and 23andMe. We show that restricting analysis to offspring of first cousins is an effective way of reducing confounding due to social/environmental correlates of FROH. Within this group in G&H+UK Biobank, we found experiment-wide significant associations between FROH and twelve common diseases. We replicated associations with type 2 diabetes (T2D) and post-traumatic stress disorder via within-sibling analysis in 23andMe (median n = 480,282). We estimated that autozygosity due to consanguinity accounts for 5%-18% of T2D cases among British Pakistanis. Our work highlights the possibility of widespread non-additive genetic effects on common diseases and has important implications for global populations with high rates of consanguinity.
Subject(s)
Consanguinity , Diabetes Mellitus, Type 2 , Humans , Diabetes Mellitus, Type 2/genetics , Homozygote , Phenotype , Polymorphism, Single Nucleotide , Biological Specimen Banks , Genome, Human , Genetic Predisposition to Disease , United KingdomABSTRACT
Schizophrenia (SCZ) is a highly heritable mental disorder with thousands of associated genetic variants located mostly in the noncoding space of the genome. Translating these associations into insights regarding the underlying pathomechanisms has been challenging because the causal variants, their mechanisms of action, and their target genes remain largely unknown. We implemented a massively parallel variant annotation pipeline (MVAP) to perform SCZ variant-to-function mapping at scale in disease-relevant neural cell types. This approach identified 620 functional variants (1.7%) that operate in a highly developmental context and neuronal-activity-dependent manner. Multimodal integration of epigenomic and CRISPRi screening data enabled us to link these functional variants to target genes, biological processes, and ultimately alterations of neuronal physiology. These results provide a multistage prioritization strategy to map functional single-nucleotide polymorphism (SNP)-to-gene-to-endophenotype relations and offer biological insights into the context-dependent molecular processes modulated by SCZ-associated genetic variation.
Subject(s)
Schizophrenia , Humans , Genetic Predisposition to Disease , Genome-Wide Association Study , Neurons/metabolism , Polymorphism, Single Nucleotide/genetics , Schizophrenia/genetics , Animals , Mice , High-Throughput Nucleotide SequencingABSTRACT
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.
Subject(s)
Multifactorial Inheritance , Quantitative Trait Loci , Humans , DNA Copy Number Variations , Phenotype , Genome, Human , Polymorphism, Single Nucleotide/geneticsABSTRACT
The 1000 Genomes Project (1kGP) is the largest fully open resource of whole-genome sequencing (WGS) data consented for public distribution without access or use restrictions. The final, phase 3 release of the 1kGP included 2,504 unrelated samples from 26 populations and was based primarily on low-coverage WGS. Here, we present a high-coverage 3,202-sample WGS 1kGP resource, which now includes 602 complete trios, sequenced to a depth of 30X using Illumina. We performed single-nucleotide variant (SNV) and short insertion and deletion (INDEL) discovery and generated a comprehensive set of structural variants (SVs) by integrating multiple analytic methods through a machine learning model. We show gains in sensitivity and precision of variant calls compared to phase 3, especially among rare SNVs as well as INDELs and SVs spanning frequency spectrum. We also generated an improved reference imputation panel, making variants discovered here accessible for association studies.
Subject(s)
Genome, Human , Whole Genome Sequencing , Female , High-Throughput Nucleotide Sequencing/methods , Humans , INDEL Mutation , Male , Polymorphism, Single NucleotideABSTRACT
Fingerprints are of long-standing practical and cultural interest, but little is known about the mechanisms that underlie their variation. Using genome-wide scans in Han Chinese cohorts, we identified 18 loci associated with fingerprint type across the digits, including a genetic basis for the long-recognized "pattern-block" correlations among the middle three digits. In particular, we identified a variant near EVI1 that alters regulatory activity and established a role for EVI1 in dermatoglyph patterning in mice. Dynamic EVI1 expression during human development supports its role in shaping the limbs and digits, rather than influencing skin patterning directly. Trans-ethnic meta-analysis identified 43 fingerprint-associated loci, with nearby genes being strongly enriched for general limb development pathways. We also found that fingerprint patterns were genetically correlated with hand proportions. Taken together, these findings support the key role of limb development genes in influencing the outcome of fingerprint patterning.
Subject(s)
Dermatoglyphics , Fingers/growth & development , Organogenesis/genetics , Polymorphism, Single Nucleotide , Toes/growth & development , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Asian People/genetics , Body Patterning/genetics , Child , Cohort Studies , Female , Forelimb/growth & development , Genetic Loci , Genome-Wide Association Study , Humans , MDS1 and EVI1 Complex Locus Protein/genetics , Male , Mice , Middle Aged , Young AdultABSTRACT
The human gut microbiota resides within a diverse chemical environment challenging our ability to understand the forces shaping this ecosystem. Here, we reveal that fitness of the Bacteroidales, the dominant order of bacteria in the human gut, is an emergent property of glycans and one specific metabolite, butyrate. Distinct sugars serve as strain-variable fitness switches activating context-dependent inhibitory functions of butyrate. Differential fitness effects of butyrate within the Bacteroides are mediated by species-level variation in Acyl-CoA thioesterase activity and nucleotide polymorphisms regulating an Acyl-CoA transferase. Using in vivo multi-omic profiles, we demonstrate Bacteroides fitness in the human gut is associated together, but not independently, with Acyl-CoA transferase expression and butyrate. Our data reveal that each strain of the Bacteroides exists within a unique fitness landscape based on the interaction of chemical components unpredictable by the effect of each part alone mediated by flexibility in the core genome.
Subject(s)
Gastrointestinal Microbiome , Metabolome , Polysaccharides/metabolism , Acyl Coenzyme A/metabolism , Amino Acid Sequence , Amino Acids, Branched-Chain/metabolism , Bacteroidetes/drug effects , Bacteroidetes/genetics , Bacteroidetes/growth & development , Butyrates/chemistry , Butyrates/pharmacology , Coenzyme A-Transferases/chemistry , Coenzyme A-Transferases/metabolism , Gastrointestinal Microbiome/drug effects , Gastrointestinal Microbiome/genetics , Genetic Variation/drug effects , Hydrogen-Ion Concentration , Metabolome/drug effects , Metabolome/genetics , Polymorphism, Single Nucleotide/genetics , Promoter Regions, Genetic/genetics , Species Specificity , Stress, Physiological/drug effects , Stress, Physiological/genetics , Transcription, Genetic/drug effectsABSTRACT
Gasdermins are a family of structurally related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while its association with genetic susceptibility to chronic mucosal inflammatory disorders is well established, little is known about its functional relevance during active disease states. Herein, we report increased GSDMB in inflammatory bowel disease, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in activated epithelial cells and organoids but instead point to increased proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display hyper-adhesiveness and enhanced formation of vinculin-based focal adhesions dependent on PDGF-A-mediated FAK phosphorylation. Importantly, carriage of disease-associated GSDMB SNPs confers functional defects, disrupting epithelial restitution/repair, which, altogether, establishes GSDMB as a critical factor for restoration of epithelial barrier function and the resolution of inflammation.
Subject(s)
Epithelial Cells/metabolism , Epithelial Cells/pathology , Inflammatory Bowel Diseases/metabolism , Inflammatory Bowel Diseases/pathology , Pore Forming Cytotoxic Proteins/metabolism , Pyroptosis , Base Sequence , Case-Control Studies , Cell Adhesion/drug effects , Cell Adhesion/genetics , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , Epithelial Cells/drug effects , Focal Adhesion Protein-Tyrosine Kinases/metabolism , HEK293 Cells , HT29 Cells , Humans , Inflammatory Bowel Diseases/genetics , Methotrexate/pharmacology , Mutation/genetics , Phosphorylation/drug effects , Polymorphism, Single Nucleotide/genetics , Pyroptosis/drug effects , Pyroptosis/genetics , Reproducibility of Results , Transcriptome/drug effects , Transcriptome/genetics , Up-Regulation/drug effects , Wound Healing/drug effects , Wound Healing/geneticsABSTRACT
Intra-tumor heterogeneity (ITH) is a mechanism of therapeutic resistance and therefore an important clinical challenge. However, the extent, origin, and drivers of ITH across cancer types are poorly understood. To address this, we extensively characterize ITH across whole-genome sequences of 2,658 cancer samples spanning 38 cancer types. Nearly all informative samples (95.1%) contain evidence of distinct subclonal expansions with frequent branching relationships between subclones. We observe positive selection of subclonal driver mutations across most cancer types and identify cancer type-specific subclonal patterns of driver gene mutations, fusions, structural variants, and copy number alterations as well as dynamic changes in mutational processes between subclonal expansions. Our results underline the importance of ITH and its drivers in tumor evolution and provide a pan-cancer resource of comprehensively annotated subclonal events from whole-genome sequencing data.
Subject(s)
Genetic Heterogeneity , Neoplasms/genetics , DNA Copy Number Variations , DNA, Neoplasm/chemistry , DNA, Neoplasm/metabolism , Databases, Genetic , Drug Resistance, Neoplasm/genetics , Humans , Neoplasms/pathology , Polymorphism, Single Nucleotide , Whole Genome SequencingABSTRACT
Osteoarthritis affects over 300 million people worldwide. Here, we conduct a genome-wide association study meta-analysis across 826,690 individuals (177,517 with osteoarthritis) and identify 100 independently associated risk variants across 11 osteoarthritis phenotypes, 52 of which have not been associated with the disease before. We report thumb and spine osteoarthritis risk variants and identify differences in genetic effects between weight-bearing and non-weight-bearing joints. We identify sex-specific and early age-at-onset osteoarthritis risk loci. We integrate functional genomics data from primary patient tissues (including articular cartilage, subchondral bone, and osteophytic cartilage) and identify high-confidence effector genes. We provide evidence for genetic correlation with phenotypes related to pain, the main disease symptom, and identify likely causal genes linked to neuronal processes. Our results provide insights into key molecular players in disease processes and highlight attractive drug targets to accelerate translation.
Subject(s)
Genetic Predisposition to Disease , Genetics, Population , Osteoarthritis/genetics , Female , Genome-Wide Association Study , Humans , Osteoarthritis/drug therapy , Phenotype , Polymorphism, Single Nucleotide/genetics , Risk Factors , Sex Characteristics , Signal Transduction/geneticsABSTRACT
3' untranslated region (3'UTR) variants are strongly associated with human traits and diseases, yet few have been causally identified. We developed the massively parallel reporter assay for 3'UTRs (MPRAu) to sensitively assay 12,173 3'UTR variants. We applied MPRAu to six human cell lines, focusing on genetic variants associated with genome-wide association studies (GWAS) and human evolutionary adaptation. MPRAu expands our understanding of 3'UTR function, suggesting that simple sequences predominately explain 3'UTR regulatory activity. We adapt MPRAu to uncover diverse molecular mechanisms at base pair resolution, including an adenylate-uridylate (AU)-rich element of LEPR linked to potential metabolic evolutionary adaptations in East Asians. We nominate hundreds of 3'UTR causal variants with genetically fine-mapped phenotype associations. Using endogenous allelic replacements, we characterize one variant that disrupts a miRNA site regulating the viral defense gene TRIM14 and one that alters PILRB abundance, nominating a causal variant underlying transcriptional changes in age-related macular degeneration.
Subject(s)
3' Untranslated Regions/genetics , Biological Evolution , Disease/genetics , Genome-Wide Association Study , Algorithms , Alleles , Gene Expression Regulation , Genes, Reporter , Genetic Variation , Humans , Phenotype , Polymorphism, Single Nucleotide/genetics , Polyribosomes/metabolism , Quantitative Trait Loci/genetics , RNA/geneticsABSTRACT
Searching for factors to improve knockin efficiency for therapeutic applications, biotechnology, and generation of non-human primate models of disease, we found that the strand exchange protein RAD51 can significantly increase Cas9-mediated homozygous knockin in mouse embryos through an interhomolog repair (IHR) mechanism. IHR is a hallmark of meiosis but only occurs at low frequencies in somatic cells, and its occurrence in zygotes is controversial. Using multiple approaches, we provide evidence for an endogenous IHR mechanism in the early embryo that can be enhanced by RAD51. This process can be harnessed to generate homozygotes from wild-type zygotes using exogenous donors and to convert heterozygous alleles into homozygous alleles without exogenous templates. Furthermore, we identify additional IHR-promoting factors and describe features of IHR events. Together, our findings show conclusive evidence for IHR in mouse embryos and describe an efficient method for enhanced gene conversion.
Subject(s)
DNA Repair/genetics , Gene Conversion , Rad51 Recombinase/metabolism , Alleles , Animals , Base Sequence , CRISPR-Associated Protein 9/metabolism , Calcium-Binding Proteins/metabolism , Cell Cycle Proteins/metabolism , Chromosomes, Mammalian/genetics , DNA Breaks, Double-Stranded , Embryo, Mammalian , Female , Genetic Loci , Homologous Recombination/genetics , Homozygote , Humans , INDEL Mutation/genetics , Mice, Inbred C57BL , Mosaicism , Nuclear Proteins/metabolism , Polymorphism, Single Nucleotide/genetics , Ribonucleoproteins/metabolism , Zygote/metabolismABSTRACT
Genetic studies have revealed many variant loci that are associated with immune-mediated diseases. To elucidate the disease pathogenesis, it is essential to understand the function of these variants, especially under disease-associated conditions. Here, we performed a large-scale immune cell gene-expression analysis, together with whole-genome sequence analysis. Our dataset consists of 28 distinct immune cell subsets from 337 patients diagnosed with 10 categories of immune-mediated diseases and 79 healthy volunteers. Our dataset captured distinctive gene-expression profiles across immune cell types and diseases. Expression quantitative trait loci (eQTL) analysis revealed dynamic variations of eQTL effects in the context of immunological conditions, as well as cell types. These cell-type-specific and context-dependent eQTLs showed significant enrichment in immune disease-associated genetic variants, and they implicated the disease-relevant cell types, genes, and environment. This atlas deepens our understanding of the immunogenetic functions of disease-associated variants under in vivo disease conditions.
Subject(s)
Gene Expression Regulation/genetics , Gene Expression/immunology , Immune System Diseases/genetics , Adult , Female , Gene Expression/genetics , Gene Expression Regulation/immunology , Genetic Predisposition to Disease/genetics , Genome-Wide Association Study/methods , Humans , Immune System/cytology , Immune System/metabolism , Immune System Diseases/metabolism , Immune System Diseases/physiopathology , Male , Middle Aged , Polymorphism, Single Nucleotide/genetics , Quantitative Trait Loci/genetics , Quantitative Trait Loci/immunology , Transcriptome/genetics , Whole Genome Sequencing/methodsABSTRACT
By following up the gut microbiome, 51 human phenotypes and plasma levels of 1,183 metabolites in 338 individuals after 4 years, we characterize microbial stability and variation in relation to host physiology. Using these individual-specific and temporally stable microbial profiles, including bacterial SNPs and structural variations, we develop a microbial fingerprinting method that shows up to 85% accuracy in classifying metagenomic samples taken 4 years apart. Application of our fingerprinting method to the independent HMP cohort results in 95% accuracy for samples taken 1 year apart. We further observe temporal changes in the abundance of multiple bacterial species, metabolic pathways, and structural variation, as well as strain replacement. We report 190 longitudinal microbial associations with host phenotypes and 519 associations with plasma metabolites. These associations are enriched for cardiometabolic traits, vitamin B, and uremic toxins. Finally, mediation analysis suggests that the gut microbiome may influence cardiometabolic health through its metabolites.
Subject(s)
Bacteria/genetics , Bacterial Proteins/metabolism , Gastrointestinal Microbiome , Metabolome , Metagenome , Microbiota , Adult , Aged , Aged, 80 and over , Bacteria/classification , Bacteria/isolation & purification , Bacteria/metabolism , Bacterial Proteins/genetics , Drug Resistance, Microbial , Feces/microbiology , Female , Genomic Instability , Humans , Longitudinal Studies , Male , Middle Aged , Phenotype , Polymorphism, Single Nucleotide , Virulence Factors/genetics , Virulence Factors/metabolism , Young AdultABSTRACT
Antibodies mediate natural and vaccine-induced immunity against viral and bacterial pathogens, whereas fungi represent a widespread kingdom of pathogenic species for which neither vaccine nor neutralizing antibody therapies are clinically available. Here, using a multi-kingdom antibody profiling (multiKAP) approach, we explore the human antibody repertoires against gut commensal fungi (mycobiota). We identify species preferentially targeted by systemic antibodies in humans, with Candida albicans being the major inducer of antifungal immunoglobulin G (IgG). Fungal colonization of the gut induces germinal center (GC)-dependent B cell expansion in extraintestinal lymphoid tissues and generates systemic antibodies that confer protection against disseminated C. albicans or C. auris infection. Antifungal IgG production depends on the innate immunity regulator CARD9 and CARD9+CX3CR1+ macrophages. In individuals with invasive candidiasis, loss-of-function mutations in CARD9 are associated with impaired antifungal IgG responses. These results reveal an important role of gut commensal fungi in shaping the human antibody repertoire through CARD9-dependent induction of host-protective antifungal IgG.
Subject(s)
Antibodies, Fungal/immunology , CARD Signaling Adaptor Proteins/metabolism , Gastrointestinal Tract/immunology , Gastrointestinal Tract/microbiology , Immunity , Immunoglobulin G/immunology , Mycobiome/immunology , Animals , B-Lymphocytes/immunology , Candida albicans/immunology , Candidiasis/immunology , Candidiasis/microbiology , Feces/microbiology , Germinal Center/immunology , Humans , Mice, Inbred C57BL , Phagocytes/metabolism , Polymorphism, Single Nucleotide/genetics , Protein Binding , Signal TransductionABSTRACT
We present evidence for multiple independent origins of recombinant SARS-CoV-2 viruses sampled from late 2020 and early 2021 in the United Kingdom. Their genomes carry single-nucleotide polymorphisms and deletions that are characteristic of the B.1.1.7 variant of concern but lack the full complement of lineage-defining mutations. Instead, the remainder of their genomes share contiguous genetic variation with non-B.1.1.7 viruses circulating in the same geographic area at the same time as the recombinants. In four instances, there was evidence for onward transmission of a recombinant-origin virus, including one transmission cluster of 45 sequenced cases over the course of 2 months. The inferred genomic locations of recombination breakpoints suggest that every community-transmitted recombinant virus inherited its spike region from a B.1.1.7 parental virus, consistent with a transmission advantage for B.1.1.7's set of mutations.
Subject(s)
COVID-19/epidemiology , COVID-19/transmission , Pandemics , Recombination, Genetic , SARS-CoV-2/genetics , Base Sequence/genetics , COVID-19/virology , Computational Biology/methods , Gene Frequency , Genome, Viral , Genotype , Humans , Mutation , Phylogeny , Polymorphism, Single Nucleotide , United Kingdom/epidemiology , Whole Genome Sequencing/methodsABSTRACT
Checkpoint inhibitors (CPIs) augment adaptive immunity. Systematic pan-tumor analyses may reveal the relative importance of tumor-cell-intrinsic and microenvironmental features underpinning CPI sensitization. Here, we collated whole-exome and transcriptomic data for >1,000 CPI-treated patients across seven tumor types, utilizing standardized bioinformatics workflows and clinical outcome criteria to validate multivariable predictors of CPI sensitization. Clonal tumor mutation burden (TMB) was the strongest predictor of CPI response, followed by total TMB and CXCL9 expression. Subclonal TMB, somatic copy alteration burden, and histocompatibility leukocyte antigen (HLA) evolutionary divergence failed to attain pan-cancer significance. Dinucleotide variants were identified as a source of immunogenic epitopes associated with radical amino acid substitutions and enhanced peptide hydrophobicity/immunogenicity. Copy-number analysis revealed two additional determinants of CPI outcome supported by prior functional evidence: 9q34 (TRAF2) loss associated with response and CCND1 amplification associated with resistance. Finally, single-cell RNA sequencing (RNA-seq) of clonal neoantigen-reactive CD8 tumor-infiltrating lymphocytes (TILs), combined with bulk RNA-seq analysis of CPI-responding tumors, identified CCR5 and CXCL13 as T-cell-intrinsic markers of CPI sensitivity.