Evolution of chromosome-arm aberrations in breast cancer through genetic network rewiring.

The basal breast cancer subtype is enriched for triple-negative breast cancer (TNBC) and displays consistent large chromosomal deletions. Here, we characterize evolution and maintenance of chromosome 4p (chr4p) loss in basal breast cancer. Analysis of The Cancer Genome Atlas data shows recurrent deletion of chr4p in basal breast cancer. Phylogenetic analysis of a panel of 23 primary tumor/patient-derived xenograft basal breast cancers reveals early evolution of chr4p deletion. Mechanistically we show that chr4p loss is associated with enhanced proliferation. Gene function studies identify an unknown gene, C4orf19, within chr4p, which suppresses proliferation when overexpressed-a member of the PDCD10-GCKIII kinase module we name PGCKA1. Genome-wide pooled overexpression screens using a barcoded library of human open reading frames identify chromosomal regions, including chr4p, that suppress proliferation when overexpressed in a context-dependent manner, implicating network interactions. Together, these results shed light on the early emergence of complex aneuploid karyotypes involving chr4p and adaptive landscapes shaping breast cancer genomes.

A cost-effective sequencing method for genetic studies combining high-depth whole exome and low-depth whole genome.

Whole genome sequencing (WGS) at high-depth (30X) allows the accurate discovery of variants in the coding and non-coding DNA regions and helps elucidate the genetic underpinnings of human health and diseases. Yet, due to the prohibitive cost of high-depth WGS, most large-scale genetic association studies use genotyping arrays or high-depth whole exome sequencing (WES). Here we propose a cost-effective method which we call "Whole Exome Genome Sequencing" (WEGS), that combines low-depth WGS and high-depth WES with up to 8 samples pooled and sequenced simultaneously (multiplexed). We experimentally assess the performance of WEGS with four different depth of coverage and sample multiplexing configurations. We show that the optimal WEGS configurations are 1.7-2.0 times cheaper than standard WES (no-plexing), 1.8-2.1 times cheaper than high-depth WGS, reach similar recall and precision rates in detecting coding variants as WES, and capture more population-specific variants in the rest of the genome that are difficult to recover when using genotype imputation methods. We apply WEGS to 862 patients with peripheral artery disease and show that it directly assesses more known disease-associated variants than a typical genotyping array and thousands of non-imputable variants per disease-associated locus.

Genomics of perivascular space burden unravels early mechanisms of cerebral small vessel disease.

Perivascular space (PVS) burden is an emerging, poorly understood, magnetic resonance imaging marker of cerebral small vessel disease, a leading cause of stroke and dementia. Genome-wide association studies in up to 40,095 participants (18 population-based cohorts, 66.3 ± 8.6 yr, 96.9% European ancestry) revealed 24 genome-wide significant PVS risk loci, mainly in the white matter. These were associated with white matter PVS already in young adults (N = 1,748; 22.1 ± 2.3 yr) and were enriched in early-onset leukodystrophy genes and genes expressed in fetal brain endothelial cells, suggesting early-life mechanisms. In total, 53% of white matter PVS risk loci showed nominally significant associations (27% after multiple-testing correction) in a Japanese population-based cohort (N = 2,862; 68.3 ± 5.3 yr). Mendelian randomization supported causal associations of high blood pressure with basal ganglia and hippocampal PVS, and of basal ganglia PVS and hippocampal PVS with stroke, accounting for blood pressure. Our findings provide insight into the biology of PVS and cerebral small vessel disease, pointing to pathways involving extracellular matrix, membrane transport and developmental processes, and the potential for genetically informed prioritization of drug targets.

Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria.

Inborn errors of human IFN-γ-dependent macrophagic immunity underlie mycobacterial diseases, whereas inborn errors of IFN-α/ß-dependent intrinsic immunity underlie viral diseases. Both types of IFNs induce the transcription factor IRF1. We describe unrelated children with inherited complete IRF1 deficiency and early-onset, multiple, life-threatening diseases caused by weakly virulent mycobacteria and related intramacrophagic pathogens. These children have no history of severe viral disease, despite exposure to many viruses, including SARS-CoV-2, which is life-threatening in individuals with impaired IFN-α/ß immunity. In leukocytes or fibroblasts stimulated in vitro, IRF1-dependent responses to IFN-γ are, both quantitatively and qualitatively, much stronger than those to IFN-α/ß. Moreover, IRF1-deficient mononuclear phagocytes do not control mycobacteria and related pathogens normally when stimulated with IFN-γ. By contrast, IFN-α/ß-dependent intrinsic immunity to nine viruses, including SARS-CoV-2, is almost normal in IRF1-deficient fibroblasts. Human IRF1 is essential for IFN-γ-dependent macrophagic immunity to mycobacteria, but largely redundant for IFN-α/ß-dependent antiviral immunity.

Epigenetic and integrative cross-omics analyses of cerebral white matter hyperintensities on MRI.

Cerebral white matter hyperintensities on MRI are markers of cerebral small vessel disease, a major risk factor for dementia and stroke. Despite the successful identification of multiple genetic variants associated with this highly heritable condition, its genetic architecture remains incompletely understood. More specifically, the role of DNA methylation has received little attention. We investigated the association between white matter hyperintensity burden and DNA methylation in blood at ∼450 000 cytosine-phosphate-guanine (CpG) sites in 9732 middle-aged to older adults from 14 community-based studies. Single CpG and region-based association analyses were carried out. Functional annotation and integrative cross-omics analyses were performed to identify novel genes underlying the relationship between DNA methylation and white matter hyperintensities. We identified 12 single CpG and 46 region-based DNA methylation associations with white matter hyperintensity burden. Our top discovery single CpG, cg24202936 (P = 7.6 × 10-8), was associated with F2 expression in blood (P = 6.4 × 10-5) and co-localized with FOLH1 expression in brain (posterior probability = 0.75). Our top differentially methylated regions were in PRMT1 and in CCDC144NL-AS1, which were also represented in single CpG associations (cg17417856 and cg06809326, respectively). Through Mendelian randomization analyses cg06809326 was putatively associated with white matter hyperintensity burden (P = 0.03) and expression of CCDC144NL-AS1 possibly mediated this association. Differentially methylated region analysis, joint epigenetic association analysis and multi-omics co-localization analysis consistently identified a role of DNA methylation near SH3PXD2A, a locus previously identified in genome-wide association studies of white matter hyperintensities. Gene set enrichment analyses revealed functions of the identified DNA methylation loci in the blood-brain barrier and in the immune response. Integrative cross-omics analysis identified 19 key regulatory genes in two networks related to extracellular matrix organization, and lipid and lipoprotein metabolism. A drug-repositioning analysis indicated antihyperlipidaemic agents, more specifically peroxisome proliferator-activated receptor-alpha, as possible target drugs for white matter hyperintensities. Our epigenome-wide association study and integrative cross-omics analyses implicate novel genes influencing white matter hyperintensity burden, which converged on pathways related to the immune response and to a compromised blood-brain barrier possibly due to disrupted cell-cell and cell-extracellular matrix interactions. The results also suggest that antihyperlipidaemic therapy may contribute to lowering risk for white matter hyperintensities possibly through protection against blood-brain barrier disruption.

Human CARMIL2 deficiency underlies a broader immunological and clinical phenotype than CD28 deficiency.

Patients with inherited CARMIL2 or CD28 deficiency have defective T cell CD28 signaling, but their immunological and clinical phenotypes remain largely unknown. We show that only one of three CARMIL2 isoforms is produced and functional across leukocyte subsets. Tested mutant CARMIL2 alleles from 89 patients and 52 families impair canonical NF-κB but not AP-1 and NFAT activation in T cells stimulated via CD28. Like CD28-deficient patients, CARMIL2-deficient patients display recalcitrant warts and low blood counts of CD4+ and CD8+ memory T cells and CD4+ TREGs. Unlike CD28-deficient patients, they have low counts of NK cells and memory B cells, and their antibody responses are weak. CARMIL2 deficiency is fully penetrant by the age of 10 yr and is characterized by numerous infections, EBV+ smooth muscle tumors, and mucocutaneous inflammation, including inflammatory bowel disease. Patients with somatic reversions of a mutant allele in CD4+ T cells have milder phenotypes. Our study suggests that CARMIL2 governs immunological pathways beyond CD28.

A systems biology approach identifies candidate drugs to reduce mortality in severely ill patients with COVID-19.

Despite the availability of highly efficacious vaccines, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lacks effective drug treatment, which results in a high rate of mortality. To address this therapeutic shortcoming, we applied a systems biology approach to the study of patients hospitalized with severe COVID. We show that, at the time of hospital admission, patients who were equivalent on the clinical ordinal scale displayed significant differential monocyte epigenetic and transcriptomic attributes between those who would survive and those who would succumb to COVID-19. We identified messenger RNA metabolism, RNA splicing, and interferon signaling pathways as key host responses overactivated by patients who would not survive. Those pathways are prime drug targets to reduce mortality of critically ill patients with COVID-19, leading us to identify tacrolimus, zotatifin, and nintedanib as three strong candidates for treatment of severely ill patients at the time of hospital admission.

PrecisionFDA Truth Challenge V2: Calling variants from short and long reads in difficult-to-map regions.

The precisionFDA Truth Challenge V2 aimed to assess the state of the art of variant calling in challenging genomic regions. Starting with FASTQs, 20 challenge participants applied their variant-calling pipelines and submitted 64 variant call sets for one or more sequencing technologies (Illumina, PacBio HiFi, and Oxford Nanopore Technologies). Submissions were evaluated following best practices for benchmarking small variants with updated Genome in a Bottle benchmark sets and genome stratifications. Challenge submissions included numerous innovative methods, with graph-based and machine learning methods scoring best for short-read and long-read datasets, respectively. With machine learning approaches, combining multiple sequencing technologies performed particularly well. Recent developments in sequencing and variant calling have enabled benchmarking variants in challenging genomic regions, paving the way for the identification of previously unknown clinically relevant variants.

Inherited human c-Rel deficiency disrupts myeloid and lymphoid immunity to multiple infectious agents.

We studied a child with severe viral, bacterial, fungal, and parasitic diseases, who was homozygous for a loss-of-function mutation of REL, encoding c-Rel, which is selectively expressed in lymphoid and myeloid cells. The patient had low frequencies of NK, effector memory cells reexpressing CD45RA (Temra) CD8+ T cells, memory CD4+ T cells, including Th1 and Th1*, Tregs, and memory B cells, whereas the counts and proportions of other leukocyte subsets were normal. Functional deficits of myeloid cells included the abolition of IL-12 and IL-23 production by conventional DC1s (cDC1s) and monocytes, but not cDC2s. c-Rel was also required for induction of CD86 expression on, and thus antigen-presenting cell function of, cDCs. Functional deficits of lymphoid cells included reduced IL-2 production by naive T cells, correlating with low proliferation and survival rates and poor production of Th1, Th2, and Th17 cytokines by memory CD4+ T cells. In naive CD4+ T cells, c-Rel is dispensable for early IL2 induction but contributes to later phases of IL2 expression. The patient's naive B cells displayed impaired MYC and BCL2L1 induction, compromising B cell survival and proliferation and preventing their differentiation into Ig-secreting plasmablasts. Inherited c-Rel deficiency disrupts the development and function of multiple myeloid and lymphoid cells, compromising innate and adaptive immunity to multiple infectious agents.

A small number of early introductions seeded widespread transmission of SARS-CoV-2 in Québec, Canada.

BACKGROUND: Québec was the Canadian province most impacted by COVID-19, with 401,462 cases as of September 24th, 2021, and 11,347 deaths due mostly to a very severe first pandemic wave. In April 2020, we assembled the Coronavirus Sequencing in Québec (CoVSeQ) consortium to sequence SARS-CoV-2 genomes in Québec to track viral introduction events and transmission within the province. METHODS: Using genomic epidemiology, we investigated the arrival of SARS-CoV-2 to Québec. We report 2921 high-quality SARS-CoV-2 genomes in the context of > 12,000 publicly available genomes sampled globally over the first pandemic wave (up to June 1st, 2020). By combining phylogenetic and phylodynamic analyses with epidemiological data, we quantify the number of introduction events into Québec, identify their origins, and characterize the spatiotemporal spread of the virus. RESULTS: Conservatively, we estimated approximately 600 independent introduction events, the majority of which happened from spring break until 2 weeks after the Canadian border closed for non-essential travel. Subsequent mass repatriations did not generate large transmission lineages (> 50 sequenced cases), likely due to mandatory quarantine measures in place at the time. Consistent with common spring break and "snowbird" destinations, most of the introductions were inferred to have originated from Europe via the Americas. Once introduced into Québec, viral lineage sizes were overdispersed, with a few lineages giving rise to most infections. Consistent with founder effects, the earliest lineages to arrive tended to spread most successfully. Fewer than 100 viral introductions arrived during spring break, of which 7-12 led to the largest transmission lineages of the first wave (accounting for 52-75% of all sequenced infections). These successful transmission lineages dispersed widely across the province. Transmission lineage size was greatly reduced after March 11th, when a quarantine order for returning travellers was enacted. While this suggests the effectiveness of early public health measures, the biggest transmission lineages had already been ignited prior to this order. CONCLUSIONS: Combined, our results reinforce how, in the absence of tight travel restrictions or quarantine measures, fewer than 100 viral introductions in a week can ensure the establishment of extended transmission chains.

Humans with inherited T cell CD28 deficiency are susceptible to skin papillomaviruses but are otherwise healthy.

We study a patient with the human papilloma virus (HPV)-2-driven "tree-man" phenotype and two relatives with unusually severe HPV4-driven warts. The giant horns form an HPV-2-driven multifocal benign epithelial tumor overexpressing viral oncogenes in the epidermis basal layer. The patients are unexpectedly homozygous for a private CD28 variant. They have no detectable CD28 on their T cells, with the exception of a small contingent of revertant memory CD4+ T cells. T cell development is barely affected, and T cells respond to CD3 and CD2, but not CD28, costimulation. Although the patients do not display HPV-2- and HPV-4-reactive CD4+ T cells in vitro, they make antibodies specific for both viruses in vivo. CD28-deficient mice are susceptible to cutaneous infections with the mouse papillomavirus MmuPV1. The control of HPV-2 and HPV-4 in keratinocytes is dependent on the T cell CD28 co-activation pathway. Surprisingly, human CD28-dependent T cell responses are largely redundant for protective immunity.

Inherited PD-1 deficiency underlies tuberculosis and autoimmunity in a child.

The pathophysiology of adverse events following programmed cell death protein 1 (PD-1) blockade, including tuberculosis (TB) and autoimmunity, remains poorly characterized. We studied a patient with inherited PD-1 deficiency and TB who died of pulmonary autoimmunity. The patient's leukocytes did not express PD-1 or respond to PD-1-mediated suppression. The patient's lymphocytes produced only small amounts of interferon (IFN)-γ upon mycobacterial stimuli, similarly to patients with inborn errors of IFN-γ production who are vulnerable to TB. This phenotype resulted from a combined depletion of Vδ2+ γδ T, mucosal-associated invariant T and CD56bright natural killer lymphocytes and dysfunction of other T lymphocyte subsets. Moreover, the patient displayed hepatosplenomegaly and an expansion of total, activated and RORγT+ CD4-CD8- double-negative αß T cells, similar to patients with STAT3 gain-of-function mutations who display lymphoproliferative autoimmunity. This phenotype resulted from excessive amounts of STAT3-activating cytokines interleukin (IL)-6 and IL-23 produced by activated T lymphocytes and monocytes, and the STAT3-dependent expression of RORγT by activated T lymphocytes. Our work highlights the indispensable role of human PD-1 in governing both antimycobacterial immunity and self-tolerance, while identifying potentially actionable molecular targets for the diagnostic and therapeutic management of TB and autoimmunity in patients on PD-1 blockade.

A coordinated progression of progenitor cell states initiates urinary tract development.

The kidney and upper urinary tract develop through reciprocal interactions between the ureteric bud and the surrounding mesenchyme. Ureteric bud branching forms the arborized collecting duct system of the kidney, while ureteric tips promote nephron formation from dedicated progenitor cells. While nephron progenitor cells are relatively well characterized, the origin of ureteric bud progenitors has received little attention so far. It is well established that the ureteric bud is induced from the nephric duct, an epithelial duct derived from the intermediate mesoderm of the embryo. However, the cell state transitions underlying the progression from intermediate mesoderm to nephric duct and ureteric bud remain unknown. Here we show that nephric duct morphogenesis results from the coordinated organization of four major progenitor cell populations. Using single cell RNA-seq and Cluster RNA-seq, we show that these progenitors emerge in time and space according to a stereotypical pattern. We identify the transcription factors Tfap2a/b and Gata3 as critical coordinators of this progenitor cell progression. This study provides a better understanding of the cellular origin of the renal collecting duct system and associated urinary tract developmental diseases, which may inform guided differentiation of functional kidney tissue.

Rare loss-of-function variants in type I IFN immunity genes are not associated with severe COVID-19.

A recent report found that rare predicted loss-of-function (pLOF) variants across 13 candidate genes in TLR3- and IRF7-dependent type I IFN pathways explain up to 3.5% of severe COVID-19 cases. We performed whole-exome or whole-genome sequencing of 1,864 COVID-19 cases (713 with severe and 1,151 with mild disease) and 15,033 ancestry-matched population controls across 4 independent COVID-19 biobanks. We tested whether rare pLOF variants in these 13 genes were associated with severe COVID-19. We identified only 1 rare pLOF mutation across these genes among 713 cases with severe COVID-19 and observed no enrichment of pLOFs in severe cases compared to population controls or mild COVID-19 cases. We found no evidence of association of rare LOF variants in the 13 candidate genes with severe COVID-19 outcomes.

Cell-free DNA tissues of origin by methylation profiling reveals significant cell, tissue, and organ-specific injury related to COVID-19 severity.

BACKGROUND: Coronavirus disease 2019 (COVID-19) primarily affects the lungs, but evidence of systemic disease with multi-organ involvement is emerging. Here, we developed a blood test to broadly quantify cell-, tissue-, and organ-specific injury due to COVID-19. METHODS: Our test leverages genome-wide methylation profiling of circulating cell-free DNA in plasma. We assessed the utility of this test to identify subjects with severe disease in two independent, longitudinal cohorts of hospitalized patients. Cell-free DNA profiling was performed on 104 plasma samples from 33 COVID-19 patients and compared to samples from patients with other viral infections and healthy controls. FINDINGS: We found evidence of injury to the lung and liver and involvement of red blood cell progenitors associated with severe COVID-19. The concentration of cell-free DNA correlated with the World Health Organization (WHO) ordinal scale for disease progression and was significantly increased in patients requiring intubation. CONCLUSIONS: This study points to the utility of cell-free DNA as an analyte to monitor and study COVID-19. FUNDING: This work was supported by NIH grants 1DP2AI138242 (to I.D.V.), R01AI146165 (to I.D.V., M.P.C., F.M.M., and J.R.), 1R01AI151059 (to I.D.V.), K08-CA230156 (to W.G.), and R33-AI129455 to C.Y.C., a Synergy award from the Rainin Foundation (to I.D.V.), a SARS-CoV-2 seed grant at Cornell (to I.D.V.), a National Sciences and Engineering Research Council of Canada fellowship PGS-D3 (to A.P.C.), and a Burroughs-Wellcome CAMS Award (to W.G.). D.C.V. is supported by a Fonds de la Recherche en Sante du Quebec Clinical Research Scholar Junior 2 award. C.Y.C. is supported by the California Initiative to Advance Precision Medicine, and the Charles and Helen Schwab Foundation.

Human T-bet Governs Innate and Innate-like Adaptive IFN-γ Immunity against Mycobacteria.

Inborn errors of human interferon gamma (IFN-γ) immunity underlie mycobacterial disease. We report a patient with mycobacterial disease due to inherited deficiency of the transcription factor T-bet. The patient has extremely low counts of circulating Mycobacterium-reactive natural killer (NK), invariant NKT (iNKT), mucosal-associated invariant T (MAIT), and Vδ2+ γδ T lymphocytes, and of Mycobacterium-non reactive classic TH1 lymphocytes, with the residual populations of these cells also producing abnormally small amounts of IFN-γ. Other lymphocyte subsets develop normally but produce low levels of IFN-γ, with the exception of CD8+ αß T and non-classic CD4+ αß TH1∗ lymphocytes, which produce IFN-γ normally in response to mycobacterial antigens. Human T-bet deficiency thus underlies mycobacterial disease by preventing the development of innate (NK) and innate-like adaptive lymphocytes (iNKT, MAIT, and Vδ2+ γδ T cells) and IFN-γ production by them, with mycobacterium-specific, IFN-γ-producing, purely adaptive CD8+ αß T, and CD4+ αß TH1∗ cells unable to compensate for this deficit.

A Distributed Whole Genome Sequencing Benchmark Study.

Population sequencing often requires collaboration across a distributed network of sequencing centers for the timely processing of thousands of samples. In such massive efforts, it is important that participating scientists can be confident that the accuracy of the sequence data produced is not affected by which center generates the data. A study was conducted across three established sequencing centers, located in Montreal, Toronto, and Vancouver, constituting Canada's Genomics Enterprise (www.cgen.ca). Whole genome sequencing was performed at each center, on three genomic DNA replicates from three well-characterized cell lines. Secondary analysis pipelines employed by each site were applied to sequence data from each of the sites, resulting in three datasets for each of four variables (cell line, replicate, sequencing center, and analysis pipeline), for a total of 81 datasets. These datasets were each assessed according to multiple quality metrics including concordance with benchmark variant truth sets to assess consistent quality across all three conditions for each variable. Three-way concordance analysis of variants across conditions for each variable was performed. Our results showed that the variant concordance between datasets differing only by sequencing center was similar to the concordance for datasets differing only by replicate, using the same analysis pipeline. We also showed that the statistically significant differences between datasets result from the analysis pipeline used, which can be unified and updated as new approaches become available. We conclude that genome sequencing projects can rely on the quality and reproducibility of aggregate data generated across a network of distributed sites.

Cell-Free DNA in Blood Reveals Significant Cell, Tissue and Organ Specific injury and Predicts COVID-19 Severity.

COVID-19 primarily affects the lungs, but evidence of systemic disease with multi-organ involvement is emerging. Here, we developed a blood test to broadly quantify cell, tissue, and organ specific injury due to COVID-19, using genome-wide methylation profiling of circulating cell-free DNA in plasma. We assessed the utility of this test to identify subjects with severe disease in two independent, longitudinal cohorts of hospitalized patients. Cell-free DNA profiling was performed on 104 plasma samples from 33 COVID-19 patients and compared to samples from patients with other viral infections and healthy controls. We found evidence of injury to the lung and liver and involvement of red blood cell progenitors associated with severe COVID-19. The concentration of cfDNA correlated with the WHO ordinal scale for disease progression and was significantly increased in patients requiring intubation. This study points to the utility of cell-free DNA as an analyte to monitor and study COVID-19.

Author Correction: Single-cell RNA-seq reveals that glioblastoma recapitulates a normal neurodevelopmental hierarchy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Single-cell RNA-seq reveals that glioblastoma recapitulates a normal neurodevelopmental hierarchy.

Cancer stem cells are critical for cancer initiation, development, and treatment resistance. Our understanding of these processes, and how they relate to glioblastoma heterogeneity, is limited. To overcome these limitations, we performed single-cell RNA sequencing on 53586 adult glioblastoma cells and 22637 normal human fetal brain cells, and compared the lineage hierarchy of the developing human brain to the transcriptome of cancer cells. We find a conserved neural tri-lineage cancer hierarchy centered around glial progenitor-like cells. We also find that this progenitor population contains the majority of the cancer's cycling cells, and, using RNA velocity, is often the originator of the other cell types. Finally, we show that this hierarchal map can be used to identify therapeutic targets specific to progenitor cancer stem cells. Our analyses show that normal brain development reconciles glioblastoma development, suggests a possible origin for glioblastoma hierarchy, and helps to identify cancer stem cell-specific targets.