Aberrant immune responses to viral pathogens contribute to pathogenesis, but our understanding of pathological immune responses caused by viruses within the human virome, especially at a population scale, remains limited. We analyzed whole-genome sequencing datasets of 6,321 Japanese individuals, including patients with autoimmune diseases (psoriasis vulgaris, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), pulmonary alveolar proteinosis (PAP) or multiple sclerosis) and coronavirus disease 2019 (COVID-19), or healthy controls. We systematically quantified two constituents of the blood DNA virome, endogenous HHV-6 (eHHV-6) and anellovirus. Participants with eHHV-6B had higher risks of SLE and PAP; the former was validated in All of Us. eHHV-6B-positivity and high SLE disease activity index scores had strong correlations. Genome-wide association study and long-read sequencing mapped the integration of the HHV-6B genome to a locus on chromosome 22q. Epitope mapping and single-cell RNA sequencing revealed distinctive immune induction by eHHV-6B in patients with SLE. In addition, high anellovirus load correlated strongly with SLE, RA and COVID-19 status. Our analyses unveil relationships between the human virome and autoimmune and infectious diseases. Analysis of the blood DNA virome in patients with COVID-19 and autoimmune disease associates endogenous HHV-6 (eHHV-6) and high anellovirus load with increased disease risk, most notably for systemic lupus erythematosus. eHHV-6 carriers show a distinct immune response.
Abstract Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1 ), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1 ), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2 ). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection.
Integration of human papillomavirus (HPV) into the host genome drives HPV-positive head and neck squamous cell carcinoma (HPV+ HNSCC). Whole-genome sequencing of 51 tumors revealed intratumor heterogeneity of HPV integration, with 44% of breakpoints subclonal, and a biased distribution of integration breakpoints across the HPV genome. Four HPV physical states were identified, with at least 49% of tumors progressing without integration. HPV integration was associated with APOBEC-induced broad genomic instability and focal genomic instability, including structural variants at integration sites. HPV+ HNSCCs exhibited almost no smoking-induced mutational signatures. Heterozygous loss of ataxia-telangiectasia mutated (ATM) was observed in 67% of tumors, with its downregulation confirmed by single-cell RNA sequencing and immunohistochemistry, suggesting ATM haploinsufficiency contributes to carcinogenesis. PI3K activation was the major oncogenic mutation, with JAK-STAT activation in tumors with clonal integration and NF-kappa B activation in those without. These findings provide valuable insights into HPV integration in HPV+ HNSCC. The integration of human papillomavirus (HPV) drives HPV-positive head and neck squamous cell carcinoma (HPV+ HNSCC). Here, the authors perform genomic analysis to investigate HPV integration in patients with HPV+ HNSCC, identifying intratumor heterogeneity and biased distribution of HPV integration.
Several X-linked genes escape from X chromosome inactivation (XCI), while differences in escape across cell types and tissues are still poorly characterized. Here, we developed scLinaX for directly quantifying relative gene expression from the inactivated X chromosome with droplet-based single-cell RNA sequencing (scRNA-seq) data. The scLinaX and differentially expressed gene analyses with large-scale blood scRNA-seq datasets consistently identified the stronger escape in lymphocytes than in myeloid cells. An extension of scLinaX to a 10x multiome dataset (scLinaX-multi) suggested a stronger escape in lymphocytes than in myeloid cells at the chromatin-accessibility level. The scLinaX analysis of human multiple-organ scRNA-seq datasets also identified the relatively strong degree of escape from XCI in lymphoid tissues and lymphocytes. Finally, effect size comparisons of genome-wide association studies between sexes suggested the underlying impact of escape on the genotype-phenotype association. Overall, scLinaX and the quantified escape catalog identified the heterogeneity of escape across cell types and tissues.
Aberrant immune responses to viral pathogens contribute to disease pathogenesis. However, the link between endogenous viruses or viral infection and autoimmune diseases is a longstanding mystery in the fields of basic and clinical science.
Objectives:
To clarify whether and which viruses comprising human viromes influence pathological immune responses and confer autoimmune disease risk. To validate clinical impacts of human virome as biomarkers of the autoimmune diseases.
Methods:
We analyzed whole-genome sequencing (WGS) datasets of human genomes of 6,321 Japanese individuals, including those with autoimmune diseases (psoriasis vulgaris [PV], rheumatoid arthritis [RA], systemic lupus erythematosus [SLE], pulmonary alveolar proteinosis [PAP], or multiple sclerosis [MS]) and severe coronavirus disease 2019 (COVID-19), or healthy controls (HC). We systematically quantified two constituents of the blood DNA virome, endogenous human herpesvirus 6A and 6B (eHHV-6A/B) integrated in the individuals' human genome sequences and anellovirus. We then conducted disease case–control association studies (Figure 1A). Replication studies were conducted using the All of Us WGS datasets (n > 240,000). Single-cell RNA sequencing (scRNA-seq) data of peripheral blood mononuclear cells (PMBCs) from the four eHHV-6B-positive patients with SLE and 11 eHHV-6B-negative patients with SLE were compared (Figure 1).
Results:
We successfully detected eHHV-6A/6B (prevalence of 0.2% and 0.1% in HC, respectively) and anellovirus (12.3%, 1.0%, and 0.29% for anellovirus-detected, -infected, and -viremic, respectively). Subjects with eHHV-6B had significantly higher risks of SLE and PAP (odds ratio (OR) = 6.9, P = 0.0061 and OR = 7.0, P = 0.0075, respectively; Figure 2A). Replication studies using the All of Us WGS datasets confirmed an elevated SLE risk of eHHV-6B (OR = 2.7, P = 0.0020 for European ancestry, and OR = 2.4, P = 0.0012 for multi-ancestry). eHHV-6B and high SLE Disease Activity Index (SLEDAI) scores had strong correlations (mean SLEDAI = 30.5 and 6.0 for eHHV-6B-positive and -negative patients with SLE, respectively; P = 1.3×10−8; Figure 2B). Single-cell RNA sequencing showed increased antiviral interferon-stimulated gene (ISG) expression profiles of monocytes in the eHHV-6B-positive patients with SLE when compared with those of the eHHV-6B-negative (Figure 2C, 2D). In addition, strong positive correlations between high anellovirus load in blood and SLE, RA, and severe COVID-19 were observed (OR = 58.5, 15.4, and 6.2 for infected status, P = 2.6×10−8, 2.5×10−5, and 0.0042, respectively).
Conclusion:
Our analyses utilizing the large-scale WGS reveal previously unknown relationships between the human virome and autoimmune and infectious diseases. Individuals carrying eHHV-6 or anellovirus positivity appears to have strikingly high impacts on disease risk and clinical phenotypes of SLE, demonstrating a value as the new informative clinical biomarker.
Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge1-5. Here we conducted a genome-wide association study (GWAS) involving 2,393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3,289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target.
Mechanisms underpinning the dysfunctional immune response in severe acute respiratory syndrome coronavirus 2 infection are elusive. We analyzed single-cell transcriptomes and T and B cell receptors (BCR) of >895,000 peripheral blood mononuclear cells from 73 coronavirus disease 2019 (COVID-19) patients and 75 healthy controls of Japanese ancestry with host genetic data. COVID-19 patients showed a low fraction of nonclassical monocytes (ncMono). We report downregulated cell transitions from classical monocytes to ncMono in COVID-19 with reduced CXCL10 expression in ncMono in severe disease. Cell-cell communication analysis inferred decreased cellular interactions involving ncMono in severe COVID-19. Clonal expansions of BCR were evident in the plasmablasts of patients. Putative disease genes identified by COVID-19 genome-wide association study showed cell type-specific expressions in monocytes and dendritic cells. A COVID-19-associated risk variant at the IFNAR2 locus (rs13050728) had context-specific and monocyte-specific expression quantitative trait loci effects. Our study highlights biological and host genetic involvement of innate immune cells in COVID-19 severity.
Abstract Human papillomavirus (HPV) integration has been considered as one of the driver factors for cancer progression, but its pattern and role in carcinogenesis is still unclear. Whole genome sequencing data of 14 Japanese HPV16+ OPSCC and 13 HPV+ OPSCC from Pan-Cancer Analysis of Whole Genomes were used to identify integrations and somatic mutations. The phylogenetic analysis of HPV16 revealed that in tumors of 14 Japanese subjects, the A4 variant was the most common (8/14, 57.1%), followed by the A5 variant (2/14, 14.3%), which is considered unique to HPV16 in East and Southeast Asian, and the A2/A3/D2/D3 variants (1/14, 7.1%), showing a significant difference from the distribution of the HPV16 sublineages in Europe and the United States, where the A1 variant accounts for half. A total of 250 integration breakpoints (BPs) in 20 of the 27 tumors (74.1%) were detected. We estimated the cancer cell fraction of integration BPs and found that HPV integration also shows intra-tumor heterogeneity, indicating that nearly half of the integration events occurred after carcinogenesis. Clonal BPs were more likely to occur in the E1 (P = 0.036), confirming that the disruption of the E1 gene may result in abnormal negative regulation of the E6/E7 oncogenes and promote OPSCC carcinogenesis. Subclonal BPs were less likely to occur in the E6 (P = 0.045), suggesting that the E6 may also play an important role in the process of cancer evolution after carcinogenesis. Four states of the HPV genome were identified: (1) episomal-only (7/27, 25.9%), (2) integrated-only (4/27, 14.8%), (3) clonally-mixed (episomal + clonally integrated) (11/27, 40.7%), and (4) subclonally-mixed (episomal + subclonally integrated) (5/27, 18.5%). Since nearly half of these cancers have developed with just episomal copies of HPV, integration itself is not essential for carcinogenesis and can occur during and after carcinogenesis. Interestingly, the E6/E7 were conserved in all four cancers with integrated-only HPV, while the E2 and E1 were disrupted or deleted. This confirms that the constitutive expression of E6/E7 is essential for the carcinogenesis of HPV+ OPSCC. These results suggest that some integrations may be drivers of carcinogenesis with disruption of E1/E2, as in these four cancers, and others may arise randomly due to genomic instability, as in the cancers with the E6/E7 deleted integrants. The number of BPs of structural variations (SVs) occurred in the overlapping 100 kb regions flanking integration BPs (ITG regions) was positively correlated with the number of integration BPs (Spearman’s rank correlation ρ = 0.78; Permutation test P = 9.9 × 10−5). The genomic instability that causes clonal integrations during carcinogenesis is considered to cause clonal SVs, mainly large duplications, and subclonal integrations and SVs such as large deletions. Clonal SVs, mainly large duplications, were also observed in the regions where only subclonal integrations occurred, suggesting that large duplications occur before integrations. Citation Format: Noah Sasa, Hirotaka Eguchi, Hidenori Tanaka, Takahito Fukusumi, Motoyuki Suzuki, Yukinori Takenaka, Yukinori Okada, Hidenori Inohara. Intra-tumor heterogeneity of HPV integration and its association with focal genomic instability in oropharyngeal cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-026.
