The genomic basis of childhood T-lineage acute lymphoblastic leukaemia.

T-lineage acute lymphoblastic leukaemia (T-ALL) is a high-risk tumour1 that has eluded comprehensive genomic characterization, which is partly due to the high frequency of noncoding genomic alterations that result in oncogene deregulation2,3. Here we report an integrated analysis of genome and transcriptome sequencing of tumour and remission samples from more than 1,300 uniformly treated children with T-ALL, coupled with epigenomic and single-cell analyses of malignant and normal T cell precursors. This approach identified 15 subtypes with distinct genomic drivers, gene expression patterns, developmental states and outcomes. Analyses of chromatin topology revealed multiple mechanisms of enhancer deregulation that involve enhancers and genes in a subtype-specific manner, thereby demonstrating widespread involvement of the noncoding genome. We show that the immunophenotypically described, high-risk entity of early T cell precursor ALL is superseded by a broader category of 'early T cell precursor-like' leukaemia. This category has a variable immunophenotype and diverse genomic alterations of a core set of genes that encode regulators of hematopoietic stem cell development. Using multivariable outcome models, we show that genetic subtypes, driver and concomitant genetic alterations independently predict treatment failure and survival. These findings provide a roadmap for the classification, risk stratification and mechanistic understanding of this disease.

Genomic landscape of Down syndrome-associated acute lymphoblastic leukemia.

Trisomy 21, the genetic cause of Down syndrome (DS), is the most common congenital chromosomal anomaly. It is associated with a 20-fold increased risk of acute lymphoblastic leukemia (ALL) during childhood and results in distinctive leukemia biology. To comprehensively define the genomic landscape of DS-ALL, we performed whole-genome sequencing and whole-transcriptome sequencing (RNA-Seq) on 295 cases. Our integrated genomic analyses identified 15 molecular subtypes of DS-ALL, with marked enrichment of CRLF2-r, IGH::IGF2BP1, and C/EBP altered (C/EBPalt) subtypes compared with 2257 non-DS-ALL cases. We observed abnormal activation of the CEBPD, CEBPA, and CEBPE genes in 10.5% of DS-ALL cases via a variety of genomic mechanisms, including chromosomal rearrangements and noncoding mutations leading to enhancer hijacking. A total of 42.3% of C/EBP-activated DS-ALL also have concomitant FLT3 point mutations or insertions/deletions, compared with 4.1% in other subtypes. CEBPD overexpression enhanced the differentiation of mouse hematopoietic progenitor cells into pro-B cells in vitro, particularly in a DS genetic background. Notably, recombination-activating gene-mediated somatic genomic abnormalities were common in DS-ALL, accounting for a median of 27.5% of structural alterations, compared with 7.7% in non-DS-ALL. Unsupervised hierarchical clustering analyses of CRLF2-rearranged DS-ALL identified substantial heterogeneity within this group, with the BCR::ABL1-like subset linked to an inferior event-free survival, even after adjusting for known clinical risk factors. These results provide important insights into the biology of DS-ALL and point to opportunities for targeted therapy and treatment individualization.

Molecular classification improves risk assessment in adult BCR-ABL1-negative B-ALL.

Genomic classification has improved risk assignment of pediatric, but not adult B-lineage acute lymphoblastic leukemia (B-ALL). The international UKALLXII/ECOG-ACRIN E2993 (#NCT00002514) trial accrued 1229 adolescent/adult patients with BCR-ABL1- B-ALL (aged 14 to 65 years). Although 93% of patients achieved remission, 41% relapsed at a median of 13 months (range, 28 days to 12 years). Five-year overall survival (OS) was 42% (95% confidence interval, 39, 44). Transcriptome sequencing, gene expression profiling, cytogenetics, and fusion polymerase chain reaction enabled genomic subtyping of 282 patient samples, of which 264 were eligible for trial, accounting for 64.5% of E2993 patients. Among patients with outcome data, 29.5% with favorable outcomes (5-year OS 65% to 80%) were deemed standard risk (DUX4-rearranged [9.2%], ETV6-RUNX1/-like [2.3%], TCF3-PBX1 [6.9%], PAX5 P80R [4.1%], high-hyperdiploid [6.9%]); 50.2% had high-risk genotypes with 5-year OS of 0% to 27% (Ph-like [21.2%], KMT2A-AFF1 [12%], low-hypodiploid/near-haploid [14.3%], BCL2/MYC-rearranged [2.8%]); 20.3% had intermediate-risk genotypes with 5-year OS of 33% to 45% (PAX5alt [12.4%], ZNF384/-like [5.1%], MEF2D-rearranged [2.8%]). IKZF1 alterations occurred in 86% of Ph-like, and TP53 mutations in patients who were low-hypodiploid (54%) and BCL2/MYC-rearranged (33%) but were not independently associated with outcome. Of patients considered high risk based on presenting age and white blood cell count, 40% harbored subtype-defining genetic alterations associated with standard- or intermediate-risk outcomes. We identified distinct immunophenotypic features for DUX4-rearranged, PAX5 P80R, ZNF384-R/-like, and Ph-like genotypes. These data in a large adult B-ALL cohort treated with a non-risk-adapted approach on a single trial show the prognostic importance of genomic analyses, which may translate into future therapeutic benefits.

Somatic and Germline Variant Calling from Next-Generation Sequencing Data.

Re-sequencing of the human genome by next-generation sequencing (NGS) has been widely applied to discover pathogenic genetic variants and/or causative genes accounting for various types of diseases including cancers. The advances in NGS have allowed the sequencing of the entire genome of patients and identification of disease-associated variants in a reasonable timeframe and cost. The core of the variant identification relies on accurate variant calling and annotation. Numerous algorithms have been developed to elucidate the repertoire of somatic and germline variants. Each algorithm has its own distinct strengths, weaknesses, and limitations due to the difference in the statistical modeling approach adopted and read information utilized. Accurate variant calling remains challenging due to the presence of sequencing artifacts and read misalignments. All of these can lead to the discordance of the variant calling results and even misinterpretation of the discovery. For somatic variant detection, multiple factors including chromosomal abnormalities, tumor heterogeneity, tumor-normal cross contaminations, unbalanced tumor/normal sample coverage, and variants with low allele frequencies add even more layers of complexity to accurate variant identification. Given the discordances and difficulties, ensemble approaches have emerged by harmonizing information from different algorithms to improve variant calling performance. In this chapter, we first introduce the general scheme of variant calling algorithms and potential challenges at distinct stages. We next review the existing workflows of variant calling and annotation, and finally explore the strategies deployed by different callers as well as their strengths and caveats. Overall, NGS-based variant identification with careful consideration allows reliable detection of pathogenic variant and candidate variant selection for precision medicine.

Genome-wide scan identifies opioid overdose risk locus close to MCOLN1.

The United States is experiencing the worst opioid overdose (OpOD) crisis in its history. We carried out a genome-wide association study on OpOD severity among 3 477 opioid-exposed individuals, 1 019 of whom experienced OpODs, including 2 032 European Americans (EAs) (653 overdose cases), and 1 445 African Americans (AAs) (366 overdose cases). Participants were scored 1 to 4 based on their reported overdose status and the number of times that medical treatment was required. Genome-wide association study (GWAS) of EAs and AAs separately resulted in two genome-wide significant (GWS) signals in AAs but none in EAs. The first signal was represented by three closely mapped variants (rs115208233, rs116181528, and rs114077267) located near mucolipin 1 (MCOLN1) and patatin-like phospholipase domain containing 6 (PNPLA6), and the other signal was represented by rs369098800 near dead-box helicase 18 (DDX18). There were no additional GWS signals in the trans-population meta-analysis, so that post-GWAS analysis focused on these loci. In network analysis, MCOLN1 was coexpressed with PNPLA6, but only MCOLN1-associated genes were enriched in functional categories relevant to OpOD, including calcium and cation channel activities; no enrichment was observed for PNPLA6-associated genes. Drug repositioning analysis was carried out in the connectivity map (CMap) database for MCOLN1 (PNPLA6 was not available in CMap) and showed that the opioid agonist drug-induced expression profile is similar to that of MCOLN1 overexpression and yielded the highest-ranked expression profile of 83 drug classes. Thus, MCOLN1 may be a risk gene for OpOD, but replication is needed. This knowledge could be helpful in the identification of drug targets for preventing OpOD.

Middle East Respiratory Syndrome Coronavirus Efficiently Infects Human Primary T Lymphocytes and Activates the Extrinsic and Intrinsic Apoptosis Pathways.

Middle East respiratory syndrome (MERS) is associated with a mortality rate of >35%. We previously showed that MERS coronavirus (MERS-CoV) could infect human macrophages and dendritic cells and induce cytokine dysregulation. Here, we further investigated the interplay between human primary T cells and MERS-CoV in disease pathogenesis. Importantly, our results suggested that MERS-CoV efficiently infected T cells from the peripheral blood and from human lymphoid organs, including the spleen and the tonsil. We further demonstrated that MERS-CoV infection induced apoptosis in T cells, which involved the activation of both the extrinsic and intrinsic apoptosis pathways. Remarkably, immunostaining of spleen sections from MERS-CoV-infected common marmosets demonstrated the presence of viral nucleoprotein in their CD3(+) T cells. Overall, our results suggested that the unusual capacity of MERS-CoV to infect T cells and induce apoptosis might partly contribute to the high pathogenicity of the virus.

Identification of TMPRSS2 as a Susceptibility Gene for Severe 2009 Pandemic A(H1N1) Influenza and A(H7N9) Influenza.

The genetic predisposition to severe A(H1N1)2009 (A[H1N1]pdm09) influenza was evaluated in 409 patients, including 162 cases with severe infection and 247 controls with mild infection. We prioritized candidate variants based on the result of a pilot genome-wide association study and a lung expression quantitative trait locus data set. The GG genotype of rs2070788, a higher-expression variant of TMPRSS2, was a risk variant (odds ratio, 2.11; 95% confidence interval, 1.18-3.77; P = .01) to severe A(H1N1)pdm09 influenza. A potentially functional single-nucleotide polymorphism, rs383510, accommodated in a putative regulatory region was identified to tag rs2070788. Luciferase assay results showed the putative regulatory region was a functional element, in which rs383510 regulated TMPRSS2 expression in a genotype-specific manner. Notably, rs2070788 and rs383510 were significantly associated with the susceptibility to A(H7N9) influenza in 102 patients with A(H7N9) influenza and 106 healthy controls. Therefore, we demonstrate that genetic variants with higher TMPRSS2 expression confer higher risk to severe A(H1N1)pdm09 influenza. The same variants also increase susceptibility to human A(H7N9) influenza.

Active replication of Middle East respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis.

Middle East respiratory syndrome coronavirus (MERS-CoV) infection caused severe pneumonia and multiorgan dysfunction and had a higher crude fatality rate (around 50% vs. 10%) than SARS coronavirus (SARS-CoV) infection. To understand the pathogenesis, we studied viral replication, cytokine/chemokine response, and antigen presentation in MERS-CoV-infected human monocyte-derived macrophages (MDMs) versus SARS-CoV-infected MDMs. Only MERS-CoV can replicate in MDMs. Both viruses were unable to significantly stimulate the expression of antiviral cytokines (interferon α [IFN-α] and IFN-ß) but induced comparable levels of tumor necrosis factor α and interleukin 6. Notably, MERS-CoV induced significantly higher expression levels of interleukin 12, IFN-γ, and chemokines (IP-10/CXCL-10, MCP-1/CCL-2, MIP-1α/CCL-3, RANTES/CCL-5, and interleukin 8) than SARS-CoV. The expression of major histocompatibility complex class I and costimulatory molecules were significantly higher in MERS-CoV-infected MDMs than in SARS-CoV-infected cells. MERS-CoV replication was validated by immunostaining of infected MDMs and ex vivo lung tissue. We conclusively showed that MERS-CoV can establish a productive infection in human macrophages. The aberrant induction of inflammatory cytokines/chemokines could be important in the disease pathogenesis.

Protocol to search for genetic factors related to severe COVID-19 by analyzing publicly available genome-wide association studies.

COVID-19 casualties vary among different ancestral groups due to a variety of factors. Here, we present a protocol for analyzing publicly available genome-wide association studies (GWASs) to search for ancestry-specific genetic factors related to severe COVID-19. We describe steps for downloading and comparing two COVID-19 GWASs, calculating expression quantitative trait loci, and single-cell gene expression analysis. We demonstrate this approach using GWASs from Host Genetics Initiative; however, it is applicable to other databases such as the UK Biobank. For complete details on the use and execution of this protocol, please refer to Cheng et al.1.

Modulating apoptosis as a novel therapeutic strategy against Respiratory Syncytial Virus infection: insights from Rotenone.

Respiratory syncytial virus (RSV) is a significant cause of acute lower respiratory tract infections, particularly in vulnerable populations such as neonates, infants, young children, and the elderly. Among infants, RSV is the primary cause of bronchiolitis and pneumonia, contributing to a notable proportion of child mortality under the age of 5. In this study, we focused on investigating the pathogenicity of a lethal RSV strain, GZ08-18, as a model for understanding mechanisms of hypervirulent RSV. Our findings indicate that the heightened pathogenicity of GZ08-18 stems from compromised activation of intrinsic apoptosis, as evidenced by aberration of mitochondrial membrane depolarization in host cells. We thus hypothesized that enhancing intrinsic apoptosis could potentially attenuate the virulence of RSV strains and explored the effects of Rotenone, a natural compound known to stimulate the intrinsic apoptosis pathway, on inhibiting RSV infection. Our results demonstrate that Rotenone treatment significantly improved mouse survival rates and mitigated lung pathology following GZ08-18 infection. These findings suggest that modulating the suppressed apoptosis induced by RSV infection represents a promising avenue for antiviral intervention strategies.

Genomic Determinants of Outcome in Acute Lymphoblastic Leukemia.

PURPOSE: Although cure rates for childhood acute lymphoblastic leukemia (ALL) exceed 90%, ALL remains a leading cause of cancer death in children. Half of relapses arise in children initially classified with standard-risk (SR) disease. MATERIALS AND METHODS: To identify genomic determinants of relapse in children with SR ALL, we performed genome and transcriptome sequencing of diagnostic and remission samples of children with SR (n = 1,381) or high-risk B-ALL with favorable cytogenetic features (n = 115) enrolled on Children's Oncology Group trials. We used a case-control study design analyzing 439 patients who relapsed and 1,057 who remained in complete remission for at least 5 years. RESULTS: Genomic subtype was associated with relapse, which occurred in approximately 50% of cases of PAX5-altered ALL (odds ratio [OR], 3.31 [95% CI, 2.17 to 5.03]; P = 3.18 × 10-8). Within high-hyperdiploid ALL, gain of chromosome 10 with disomy of chromosome 7 was associated with favorable outcome (OR, 0.27 [95% CI, 0.17 to 0.42]; P = 8.02 × 10-10; St Jude Children's Research Hospital validation cohort: OR, 0.22 [95% CI, 0.05 to 0.80]; P = .009), and disomy of chromosomes 10 and 17 with gain of chromosome 6 was associated with relapse (OR, 7.16 [95% CI, 2.63 to 21.51]; P = 2.19 × 10-5; validation cohort: OR, 21.32 [95% CI, 3.62 to 119.30]; P = .0004). Genomic alterations were associated with relapse in a subtype-dependent manner, including alterations of INO80 in ETV6::RUNX1 ALL, IKZF1, and CREBBP in high-hyperdiploid ALL and FHIT in BCR::ABL1-like ALL. Genomic alterations were also associated with the presence of minimal residual disease, including NRAS and CREBBP in high-hyperdiploid ALL. CONCLUSION: Genetic subtype, patterns of aneuploidy, and secondary genomic alterations determine risk of relapse in childhood ALL. Comprehensive genomic analysis is required for optimal risk stratification.

Biologic and clinical analysis of childhood gamma delta T-ALL identifies LMO2/STAG2 rearrangements as extremely high-risk.

Acute lymphoblastic leukemia expressing the gamma delta T cell receptor (yo T-ALL) is a poorly understood disease. We studied 200 children with yo T-ALL from 13 clinical study groups to understand the clinical and genetic features of this disease. We found age and genetic drivers were significantly associated with outcome. yo T-ALL diagnosed in children under three years of age was extremely high-risk and enriched for genetic alterations that result in both LMO2 activation and STAG2 inactivation. Mechanistically, using patient samples and isogenic cell lines, we show that inactivation of STAG2 profoundly perturbs chromatin organization by altering enhancer-promoter looping, resulting in deregulation of gene expression associated with T-cell differentiation. High throughput drug screening identified a vulnerability in DNA repair pathways arising from STAG2 inactivation, which can be targeted by Poly(ADP-ribose) polymerase (PARP) inhibition. These data provide a diagnostic framework for classification and risk stratification of pediatric yo T-ALL.

The effect of gamma sterilization on the insecticidal toxicity of engineered and conventional Bacillus thuringiensis strains.

This study evaluates the effect of gamma radiation on the spore activity, toxicity, and crystal structures of two engineered Bacillus thuringiensis (Bt) strains, TnX and TnY, and the reference Bt strain HD-1. We attempted to identify dosages of cobalt-60 gamma radiation that would inactivate Bt spores but not affect its toxicity. In the radiation dosage range of 10-15 kilogray, no viable spore formation and no significant reduction of the efficiency of Bt against lepidopteran larvae were observed. However, further sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results show that the components of the protoxin are affected by gamma radiation and that some bands are absent after treatment compared with the controls; the change in the protoxin band pattern depends on the type of Bt strain. Furthermore, the spore crystal structure of three Bt strains was studied with scanning electron microscopy and transmission electron microscopy. The results show that there are no changes in the size or shape of the treated Bt spores and crystals compared with the controls. The use of gamma radiation is effective to inactivate the spores of engineered Bt strains while preserving stable Bt toxicity against the target insect larvae.

A functional variation in CD55 increases the severity of 2009 pandemic H1N1 influenza A virus infection.

Infection due to 2009 pandemic H1N1 influenza A virus (A[H1N1]pdm09) is commonly manifested as mild infection but occasionally as severe pneumonia. We hypothesized that host genetic variations may contribute to disease severity. An initially small-scale genome-wide association study guided the selection of CD55 single-nucleotide polymorphisms in 425 Chinese patients with severe (n = 177) or mild (n = 248) disease. Carriers of rs2564978 genotype T/T were significantly associated with severe infection (odds ratio, 1.75; P = .011) under a recessive model, after adjustment for clinical confounders. An allele-specific effect on CD55 expression was revealed and ascribed to a promoter indel variation, which was in complete linkage disequilibrium with rs2564978. The promoter variant with deletion exhibited significantly lower transcriptional activity. We further demonstrated that CD55 can protect respiratory epithelial cells from complement attack. Additionally, A(H1N1)pdm09 infection promoted CD55 expression. In conclusion, CD55 polymorphisms are associated with severe A(H1N1)pdm09 infection. CD55 may exert a substantial impact on the disease severity of A(H1N1)pdm09 infection.

MAP3K19 regulatory variation in populations with African ancestry may increase COVID-19 severity.

To identify ancestry-linked genetic risk variants associated with COVID-19 hospitalization, we performed an integrative analysis of two genome-wide association studies and resolved four single nucleotide polymorphisms more frequent in COVID-19-hospitalized patients with non-European ancestry. Among them, the COVID-19 risk SNP rs16831827 shows the largest difference in minor allele frequency (MAF) between populations with African and European ancestry and also shows higher MAF in hospitalized COVID-19 patients among cohorts of mixed ancestry (odds ratio [OR] = 1.20, 95% CI: 1.10-1.30) and entirely African ancestry (OR = 1.30, 95% CI: 1.02-1.67). rs16831827 is an expression quantitative trait locus of MAP3K19. MAP3K19 expression is induced during ciliogenesis and most abundant in ciliated tissues including lungs. Single-cell RNA sequencing analyses revealed that MAP3K19 is highly expressed in multiple ciliated cell types. As rs16831827∗T is associated with reduced MAP3K19 expression, it may increase the risk of severe COVID-19 by reducing MAP3K19 expression.

Regulatory variants of APOBEC3 genes potentially associate with COVID-19 severity in populations with African ancestry.

Since November 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused the worldwide pandemic of the coronavirus disease 2019 (COVID-19), the impact of which is huge to the lives of world populations. Many studies suggested that such situation will continue due to the endless mutations in SARS-CoV-2 genome that result in complexity of the efforts for the control of SARS-CoV-2, since the special enrichment of nucleotide substitution C>U in SARS-CoV-2 sequences were discovered mainly due to the editing by human host factors APOBEC3 genes. The observation of SARS-CoV-2 variants Beta (B.1.351) and Omicron (B.1.1.529) firstly spreading in South Africa promoted us to hypothesize that genetic variants of APOBEC3 special in African populations may be attributed to the higher mutation rate of SARS-CoV-2 variants in Africa. Current study was conducted to search for functional variants of APOBEC3 genes associate with COVID-19 hospitalization in African population. By integrating data from the 1000 Genomes Project, Genotype-Tissue Expression (GTEx), and Host Genetics Initiative (HGI) of COVID-19, we identified potential functional SNPs close to APOBEC3 genes that are associated with COVID-19 hospitalization in African but not with other populations. Our study provides new insights on the potential contribution of APOBEC3 genes on the evolution of SARS-CoV-2 mutations in African population, but further replication is needed to confirm our results.

The utility of camptothecin as a synergist of Bacillus thuringiensis var. kurstaki and nucleopolyhedroviruses against Trichoplusia ni and Spodoptera exigua.

We studied the effect of combining microbial pesticides with camptothecin (CPT) on the mortality of two lepidopteran insects: Trichoplusia ni (Hübner) and Spodoptera exigua (Hübner). CPT is an alkaloid that is often used as an anticancer agent. Here, CPT was evaluated as a microbial pesticide synergist of Bacillus thuringiensis (Bt) and insect baculovirus. The toxicity of CPT and its synergistic effects on two microbial pesticides were studied using the diet overlay method. Bioassay results showed that CPT significantly enhances the toxicity of Bt variety kurstaki to S. exigua and T ni. In addition, CPT strongly enhanced the infectivity of Autographa californica (Speyer) multinucleocapsid nucleopolyhedrovirus (AcMNPV) and S. exigua nucleopolyhedrovirus (SeMNPV). Using light microscopy, we found that CPT disrupts the peritrophic membrane of T. ni larvae and severely affects the structure of the midgut, resulting in an abnormal gut lumen morphology. We speculate that CPT increases toxicity by affecting the permeability of the peritrophic membrane.

Genome-wide screening of sex-biased genetic variants potentially associated with COVID-19 hospitalization.

Sex-biased difference in coronavirus disease 2019 (COVID-19) hospitalization has been observed as that male patients tend to be more likely to be hospitalized than female patients. However, due to the insufficient sample size and existed studies that more prioritized to sex-stratified COVID-19 genome-wide association study (GWAS), the searching for sex-biased genetic variants showing differential association signals between sexes with COVID-19 hospitalization was severely hindered. We hypothesized genetic variants would show potentially sex-biased genetic effects on COVID-19 hospitalization if they display significant differential association effect sizes between male and female COVID-19 patients. By integrating two COVID-19 GWASs, including hospitalized COVID-19 patients vs. general population separated into males (case = 1,917 and control = 221,174) and females (case = 1,343 and control = 262,886), we differentiated the association effect sizes of each common single nucleotide polymorphism (SNP) within the two GWASs. Twelve SNPs were suggested to show differential COVID-19 associations between sexes. Further investigation of genes (n = 58) close to these 12 SNPs resulted in the identification of 34 genes demonstrating sex-biased differential expression in at least one GTEx tissue. Finally, 5 SNPs are mapped to 8 genes, including rs1134004 (GADD45G), rs140657166 (TRIM29 and PVRL1), rs148143613 (KNDC1 and STK32C), rs2443615 (PGAP2 and TRIM21), and rs2924725 (CSMD1). The 8 genes display significantly differential gene expression in blood samples derived from COVID-19 patients compared to healthy controls. These genes are potential genetic factors contributing to sex differences in COVID-19 hospitalization and warranted for further functional studies.

Epigenome-wide association study of posttraumatic stress disorder identifies novel loci in U.S. military veterans.

Posttraumatic stress disorder (PTSD) is a chronic and disabling psychiatric disorder prevalent in military veterans. Epigenetic mechanisms have been implicated in the etiology of PTSD, with DNA methylation being the most studied to identify novel molecular biomarkers associated with this disorder. We performed one of the largest single-sample epigenome-wide association studies (EWAS) of PTSD to date. Our sample included 1135 male European-American U.S. veterans who participated in the National Health and Resilience in Veterans Study (NHRVS). DNA was collected from saliva samples and the Illumina HumanMethylation EPIC BeadChip was used for the methylation analysis. PTSD was assessed using the PTSD Checklist. An EWAS was conducted using linear regression adjusted for age, cell-type proportions, first 10 principal components, and smoking status. After Bonferroni correction, we identified six genome-wide significant (GWS) CpG sites associated with past-month PTSD and three CpGs with lifetime PTSD (prange = 10-10-10-8). These CpG sites map to genes involved in immune function, transcription regulation, axonal guidance, cell signaling, and protein binding. Among these, SENP7, which is involved in transcription regulation and has been linked to risk-taking behavior and alcohol consumption in genome-wide association studies, replicated in an independent veteran cohort and was downregulated in medial orbitofrontal cortex of PTSD postmortem brain tissue. These findings suggest potential epigenetic biomarkers of PTSD that may help inform the pathophysiology of this disorder in veterans and other trauma-affected populations.