Multiomic analysis of uterine leiomyomas in self-described Black and White women: molecular insights into health disparities.

BACKGROUND: Black women are at an increased risk of developing uterine leiomyomas and experiencing worse disease prognosis than White women. Epidemiologic and molecular factors have been identified as underlying these disparities, but there remains a paucity of deep, multiomic analysis investigating molecular differences in uterine leiomyomas from Black and White patients. OBJECTIVE: To identify molecular alterations within uterine leiomyoma tissues correlating with patient race by multiomic analyses of uterine leiomyomas collected from cohorts of Black and White women. STUDY DESIGN: We performed multiomic analysis of uterine leiomyomas from Black (42) and White (47) women undergoing hysterectomy for symptomatic uterine leiomyomata. In addition, our analysis included the application of orthogonal methods to evaluate fibroid biomechanical properties, such as second harmonic generation microscopy, uniaxial compression testing, and shear-wave ultrasonography analyses. RESULTS: We found a greater proportion of MED12 mutant uterine leiomyomas from Black women (>35% increase; Mann-Whitney U, P<.001). MED12 mutant tumors exhibited an elevated abundance of extracellular matrix proteins, including several collagen isoforms, involved in the regulation of the core matrisome. Histologic analysis of tissue fibrosis using trichrome staining and secondary harmonic generation microscopy confirmed that MED12 mutant tumors are more fibrotic than MED12 wild-type tumors. Using shear-wave ultrasonography in a prospectively collected cohort, Black patients had fibroids that were firmer than White patients, even when similar in size. In addition, these analyses uncovered ancestry-linked expression quantitative trait loci with altered allele frequencies in African and European populations correlating with differential abundance of several proteins in uterine leiomyomas independently of MED12 mutation status, including tetracoidpeptide repeat protein 38. CONCLUSION: Our study shows that Black women have a higher prevalence of uterine leiomyomas harboring mutations in MED12 and that this mutational status correlates with increased tissue fibrosis compared with wild-type uterine leiomyomas. Our study provides insights into molecular alterations correlating with racial disparities in uterine leiomyomas and improves our understanding of the molecular etiology underlying uterine leiomyoma development within these populations.

Proteogenomic analysis of enriched HGSOC tumor epithelium identifies prognostic signatures and therapeutic vulnerabilities.

We performed a deep proteogenomic analysis of bulk tumor and laser microdissection enriched tumor cell populations from high-grade serous ovarian cancer (HGSOC) tissue specimens spanning a broad spectrum of purity. We identified patients with longer progression-free survival had increased immune-related signatures and validated proteins correlating with tumor-infiltrating lymphocytes in 65 tumors from an independent cohort of HGSOC patients, as well as with overall survival in an additional 126 HGSOC patient cohort. We identified that homologous recombination deficient (HRD) tumors are enriched in pathways associated with metabolism and oxidative phosphorylation that we validated in independent patient cohorts. We further identified that polycomb complex protein BMI-1 is elevated in HR proficient (HRP) tumors, that elevated BMI-1 correlates with poor overall survival in HRP but not HRD HGSOC patients, and that HRP HGSOC cells are uniquely sensitive to BMI-1 inhibition.

Transcriptomic Profiling and Pathway Analysis of Mesenchymal Stem Cells Following Low Dose-Rate Radiation Exposure.

Low dose-rate radiation exposure can occur in medical imaging, as background from environmental or industrial radiation, and is a hazard of space travel. In contrast with high dose-rate radiation exposure that can induce acute life-threatening syndromes, chronic low-dose radiation is associated with Chronic Radiation Syndrome (CRS), which can alter environmental sensitivity. Secondary effects of chronic low dose-rate radiation exposure include circulatory, digestive, cardiovascular, and neurological diseases, as well as cancer. Here, we investigated 1-2 Gy, 0.66 cGy/h, 60Co radiation effects on primary human mesenchymal stem cells (hMSC). There was no significant induction of apoptosis or DNA damage, and cells continued to proliferate. Gene ontology (GO) analysis of transcriptome changes revealed alterations in pathways related to cellular metabolism (cholesterol, fatty acid, and glucose metabolism), extracellular matrix modification and cell adhesion/migration, and regulation of vasoconstriction and inflammation. Interestingly, there was increased hypoxia signaling and increased activation of pathways regulated by iron deficiency, but Nrf2 and related genes were reduced. The data were validated in hMSC and human lung microvascular endothelial cells using targeted qPCR and Western blotting. Notably absent in the GO analysis were alteration pathways for DNA damage response, cell cycle inhibition, senescence, and pro-inflammatory response that we previously observed for high dose-rate radiation exposure. Our findings suggest that cellular gene transcription response to low dose-rate ionizing radiation is fundamentally different compared to high-dose-rate exposure. We hypothesize that cellular response to hypoxia and iron deficiency are driving processes, upstream of the other pathway regulation.

Proteogenomic analysis of lung adenocarcinoma reveals tumor heterogeneity, survival determinants, and therapeutically relevant pathways.

We present a deep proteogenomic profiling study of 87 lung adenocarcinoma (LUAD) tumors from the United States, integrating whole-genome sequencing, transcriptome sequencing, proteomics and phosphoproteomics by mass spectrometry, and reverse-phase protein arrays. We identify three subtypes from somatic genome signature analysis, including a transition-high subtype enriched with never smokers, a transversion-high subtype enriched with current smokers, and a structurally altered subtype enriched with former smokers, TP53 alterations, and genome-wide structural alterations. We show that within-tumor correlations of RNA and protein expression associate with tumor purity and immune cell profiles. We detect and independently validate expression signatures of RNA and protein that predict patient survival. Additionally, among co-measured genes, we found that protein expression is more often associated with patient survival than RNA. Finally, integrative analysis characterizes three expression subtypes with divergent mutations, proteomic regulatory networks, and therapeutic vulnerabilities. This proteogenomic characterization provides a foundation for molecularly informed medicine in LUAD.

APOE Is Associated With Serum Tau Following Uncomplicated Mild Traumatic Brain Injury.

Background and Objectives: APOE e4 has been linked to poor outcome following traumatic brain injury (TBI); however, the mechanisms behind this relationship are unclear. Few studies have investigated the relationship between the APOE genotype and established brain related protein biomarkers following TBI. The purpose of this study was to examine this relationship in service members and veterans (SMVs) following TBI. Methods: Participants were 209 SMVs [124 uncomplicated mild TBI (mTBI); 85 complicated mild, moderate, severe, or penetrating TBI (mod-sev TBI)] prospectively enrolled in the DVBIC-TBICoE 15-Year Longitudinal TBI Study. APOE genotyping was undertaken using non-fasting blood serum samples. Participants were divided into three groups: APOE e2+, APOE e3/e3, and APOE e4+. Results: In participants with mTBI, those with the APOE e2 allele had significantly lower levels of tau than those with APOE e4 (p = 0.005, r = 0.43, medium-large effect size). Those with APOE e3/e3 trended toward having higher tau than those APOE e2+ (p = 0.076, r = 0.20, small-medium effect size) and lower tau than those with APOE e4+ (p = 0.062, r = 0.21, small-medium effect size). There were no significant differences in biomarkers based on APOE in the mod-sev TBI group. Discussion: This study is the first to demonstrate APOE genotype is related to serum tau levels following a mTBI, extending prior findings to human serum following mTBI. In addition to higher serum tau levels in APOE e4 carriers, lower tau levels were observed in APOE e2 carriers, suggesting a possible protective effect.

Peptide ancestry informative markers in uterine neoplasms from women of European, African, and Asian ancestry.

Characterization of ancestry-linked peptide variants in disease-relevant patient tissues represents a foundational step to connect patient ancestry with disease pathogenesis. Nonsynonymous single-nucleotide polymorphisms encoding missense substitutions within tryptic peptides exhibiting high allele frequencies in European, African, and East Asian populations, termed peptide ancestry informative markers (pAIMs), were prioritized from 1000 genomes. In silico analysis identified that as few as 20 pAIMs can determine ancestry proportions similarly to >260K SNPs (R2 = 0.99). Multiplexed proteomic analysis of >100 human endometrial cancer cell lines and uterine leiomyoma tissues combined resulted in the quantitation of 62 pAIMs that correlate with patient race and genotype-confirmed ancestry. Candidates include a D451E substitution in GC vitamin D-binding protein previously associated with altered vitamin D levels in African and European populations. pAIMs will support generalized proteoancestry assessment as well as efforts investigating the impact of ancestry on the human proteome and how this relates to the pathogenesis of uterine neoplasms.

Recruitment of MLL1 complex is essential for SETBP1 to induce myeloid transformation.

Abnormal activation of SETBP1 due to overexpression or missense mutations occurs frequently in various myeloid neoplasms and associates with poor prognosis. Direct activation of Hoxa9/Hoxa10/Myb transcription by SETBP1 and its missense mutants is essential for their transforming capability; however, the underlying epigenetic mechanisms remain elusive. We found that both SETBP1 and its missense mutant SETBP1(D/N) directly interact with histone methyltransferase MLL1. Using a combination of ChIP-seq and RNA-seq analysis in primary hematopoietic stem and progenitor cells, we uncovered extensive overlap in their genomic occupancy and their cooperation in activating many oncogenic transcription factor genes including Hoxa9/Hoxa10/Myb and a large group of ribosomal protein genes. Genetic ablation of Mll1 as well as treatment with an inhibitor of the MLL1 complex OICR-9429 abrogated Setbp1/Setbp1(D/N)-induced transcriptional activation and transformation. Thus, the MLL1 complex plays a critical role in Setbp1-induced transcriptional activation and transformation and represents a promising target for treating myeloid neoplasms with SETBP1 activation.

Rare variant association study of veteran twin whole-genomes links severe depression with a nonsynonymous change in the neuronal gene BHLHE22.

OBJECTIVES: Major Depressive Disorder (MDD) is a complex neuropsychiatric disease with known genetic associations, but without known links to rare variation in the human genome. Here we aim to identify rare genetic variants associated with MDD using deep whole-genome sequencing data in an independent population. METHODS: We report the sequencing of 1,688 whole genomes in a large sample of male-male Veteran twins. Depression status was classified based on a structured diagnostic interview according to DSM-III-R diagnostic criteria. Searching only rare variants in genomic regions from recent GWAS on MDD, we used the optimised sequence kernel association test and Fisher's Exact test to fine map loci associated with severe depression. RESULTS: Our analysis identified one gene associated with severe depression, basic helix loop helix e22 (PAdjusted = 0.03) via SKAT-O test between unrelated severely depressed cases compared to unrelated non-depressed controls. The same gene BHLHE22 had a non-silent variant rs13279074 (PAdjusted = 0.032) based on a single variant Fisher's Exact test between unrelated severely depressed cases compared to unrelated non-depressed controls. CONCLUSION: The gene BHLHE22 shows compelling genetic evidence of directly impacting the severe depression phenotype. Together these results advance understanding of the genetic contribution to major depressive disorder in a new cohort and link a rare variant to severe forms of the disorder.

Transcriptomic profiling and pathway analysis of cultured human lung microvascular endothelial cells following ionizing radiation exposure.

The vascular system is sensitive to radiation injury, and vascular damage is believed to play a key role in delayed tissue injury such as pulmonary fibrosis. However, the response of endothelial cells to radiation is not completely understood. We examined the response of primary human lung microvascular endothelial cells (HLMVEC) to 10 Gy (1.15 Gy/min) X-irradiation. HLMVEC underwent senescence (80-85%) with no significant necrosis or apoptosis. Targeted RT-qPCR showed increased expression of genes CDKN1A and MDM2 (10-120 min). Western blotting showed upregulation of p2/waf1, MDM2, ATM, and Akt phosphorylation (15 min-72 h). Low levels of apoptosis at 24-72 h were identified using nuclear morphology. To identify novel pathway regulation, RNA-seq was performed on mRNA using time points from 2 to 24 h post-irradiation. Gene ontology and pathway analysis revealed increased cell cycle inhibition, DNA damage response, pro- and anti- apoptosis, and pro-senescence gene expression. Based on published literature on inflammation and endothelial-to-mesenchymal transition (EndMT) pathway genes, we identified increased expression of pro-inflammatory genes and EndMT-associated genes by 24 h. Together our data reveal a time course of integrated gene expression and protein activation leading from early DNA damage response and cell cycle arrest to senescence, pro-inflammatory gene expression, and endothelial-to-mesenchymal transition.

Apolipoprotein e (APOE) ε4 genotype influences memory performance following remote traumatic brain injury in U.S. military service members and veterans.

The purpose of this study was to examine the association between the apolipoprotein E (APOE) ε4 allele and neurocognitive functioning following traumatic brain injury (TBI) in military service members and veterans (SMVs). Participants included 176 SMVs with a history of remote TBI (≥1 year post-injury), categorized into mild (n = 100), moderate (n = 40), and severe (n = 36) TBI groups. Participants completed a neuropsychological assessment and APOE genotyping (n = 46 ε4+, n = 130 ε4-). Neurocognitive composite scores representing memory, executive functioning, and visual processing speed were computed. ANCOVAs adjusting for race, education, combat exposure, and PTSD symptom severity showed a significant main effect of ε4 on the memory composite, such that ε4+ SMVs exhibited poorer memory performance than ε4- SMVs. When ε2 allele carriers were removed from the analyses, associations with memory were strengthened, demonstrating a possible protective effect of the ε2 allele. No main effect of TBI group was identified on any cognitive composite, nor were there any significant TBI group × Îµ4 status interactions for any cognitive composite. Future studies with larger samples are needed to verify these findings, but our results suggest an important relationship between ε4 status and memory functioning following remote TBI of all severities.

Apolipoprotein E e4 is associated with worse self-reported neurobehavioral symptoms following uncomplicated mild traumatic brain injury in U.S. military service members.

Past research has found a relationship between the apolipoprotein E (APOE) e4 allele and worse neurobehavioral functioning following mild traumatic brain injury (MTBI) in civilian populations. The purpose of this study was to examine this relationship in service members and veterans (SMVs) following MTBI. Participants were 151 SMVs (103 uncomplicated MTBI; 48 Injured Controls [IC]) prospectively enrolled in the DVBIC-TBICoE 15-Year Longitudinal TBI Study. Participants completed a battery of self-reported neurobehavioral symptom measures on average 76.2 months post-injury (SD = 31.8). APOE genotyping was undertaken using non-fasting blood samples. Participants were classified into four subgroups based on injury (MTBI vs. IC) and APOE e4 allele status (e4 present/absent). In the IC group, there were no significant differences across APOE e4 status subgroups for all measures. In the MTBI group, participants with the APOE e4 allele had significantly worse scores on measures of depression, pain, anxiety, grief, positive well-being, social participation, and resilience compared to those without the e4 allele (d = .44 to d = .69). When comparing the number of 'clinically elevated' neurobehavioral measures simultaneously, the MTBI/e4 present subgroup consistently had a higher number of elevated measures compared to the MTBI/e4 absent, IC/e4 present, and IC/e4 absent subgroups. The APOE e4 allele was associated with poorer neurobehavioral outcome in SMVs in the chronic phase of recovery following MTBI. APOE e4 could be incorporated into screening tools to predict SMVs at risk for poor long-term neurobehavioral outcome in an effort to provide early intervention to improve long-term clinical outcome.

Personalized Single-Cell Proteogenomics to Distinguish Acute Myeloid Leukemia from Non-Malignant Clonal Hematopoiesis.

Genetic mutations associated with acute myeloid leukemia (AML) also occur in age-related clonal hematopoiesis, often in the same individual. This makes confident assignment of detected variants to malignancy challenging. The issue is particularly crucial for AML post-treatment measurable residual disease monitoring, where results can be discordant between genetic sequencing and flow cytometry. We show here, that it is possible to distinguish AML from clonal hematopoiesis and to resolve the immunophenotypic identity of clonal architecture. To achieve this, we first design patient-specific DNA probes based on patient's whole-genome sequencing, and then use them for patient-personalized single-cell DNA sequencing with simultaneous single-cell antibody-oligonucleotide sequencing. Examples illustrate AML arising from DNMT3A and TET2 mutated clones as well as independently. The ability to personalize single-cell proteogenomic assessment for individual patients based on leukemia-specific genomic features has implications for ongoing AML precision medicine efforts.

Tensin 1 (TNS1) is a modifier gene for low body mass index (BMI) in homozygous [F508del]CFTR patients.

Cystic fibrosis (CF) is a life-limiting autosomal recessive genetic disease caused by variants in the CFTR gene, most commonly by the [F508del] variant. Although CF is a classical Mendelian disease, genetic variants in several modifier genes have been associated with variation of the clinical phenotype for pulmonary and gastrointestinal function and urogenital development. We hypothesized that whole genome sequencing of a well-phenotyped CF populations might identify novel variants in known, or hitherto unknown, modifier genes. Whole genome sequencing was performed on the Illumina HiSeq X platform for 98 clinically diagnosed cystic fibrosis patient samples from the Adult CF Clinic at the University of California San Diego (UCSD). We compared protein-coding, non-silent variants genome wide between CFTR [F508del] homozygotes vs CFTR compound heterozygotes. Based on a single variant score test, we found 3 SNPs in common variants (MAF >5%) that occurred at significantly different rates between homozygous [F508del]CFTR and compound heterozygous [F508del]CFTR patients. The 3 SNPs were all located in one gene on chromosome 2: Tensin 1 (TNS1: rs3796028; rs2571445: and rs918949). We observed significantly lower BMIs in homozygous [F508del]CFTR patients who were also homozygous for Tensin 1 rs918949 (T/T) (p = 0.023) or rs2571445 (G/G) (p = 0.02) variants. The Tensin 1 gene is thus a potential modifier gene for low BMI in CF patients homozygous for the [F508del]CFTR variant.

Survival motor neuron deficiency slows myoblast fusion through reduced myomaker and myomixer expression.

BACKGROUND: Spinal muscular atrophy is an inherited neurodegenerative disease caused by insufficient levels of the survival motor neuron (SMN) protein. Recently approved treatments aimed at increasing SMN protein levels have dramatically improved patient survival and have altered the disease landscape. While restoring SMN levels slows motor neuron loss, many patients continue to have smaller muscles and do not achieve normal motor milestones. While timing of treatment is important, it remains unclear why SMN restoration is insufficient to fully restore muscle size and function. We and others have shown that SMN-deficient muscle precursor cells fail to efficiently fuse into myotubes. However, the role of SMN in myoblast fusion is not known. METHODS: In this study, we show that SMN-deficient myoblasts readily fuse with wild-type myoblasts, demonstrating fusion competency. Conditioned media from wild type differentiating myoblasts do not rescue the fusion deficit of SMN-deficient cells, suggesting that compromised fusion may primarily be a result of altered membrane dynamics at the cell surface. Transcriptome profiling of skeletal muscle from SMN-deficient mice revealed altered expression of cell surface fusion molecules. Finally, using cell and mouse models, we investigate if myoblast fusion can be rescued in SMN-deficient myoblast and improve the muscle pathology in SMA mice. RESULTS: We found reduced expression of the muscle fusion proteins myomaker (P = 0.0060) and myomixer (P = 0.0051) in the muscle of SMA mice. Suppressing SMN expression in C2C12 myoblast cells reduces expression of myomaker (35% reduction; P < 0.0001) and myomixer, also known as myomerger and minion, (30% reduction; P < 0.0001) and restoring SMN levels only partially restores myomaker and myomixer expression. Ectopic expression of myomixer improves myofibre number (55% increase; P = 0.0006) and motor function (35% decrease in righting time; P = 0.0089) in SMA model mice and enhances motor function (82% decrease in righting time; P < 0.0001) and extends survival (28% increase; P < 0.01) when administered in combination with an antisense oligonucleotide that increases SMN protein levels. CONCLUSIONS: Here, we identified reduced expression of muscle fusion proteins as a key factor in the fusion deficits of SMN-deficient myoblasts. This discovery provides a novel target to improve SMA muscle pathology and motor function, which in combination with SMN increasing therapy could enhance clinical outcomes for SMA patients.

Proteogenomic landscape of uterine leiomyomas from hereditary leiomyomatosis and renal cell cancer patients.

Pathogenic mutations in fumarate hydratase (FH) drive hereditary leiomyomatosis and renal cell cancer (HLRCC) and increase the risk of developing uterine leiomyomas (ULMs). An integrated proteogenomic analysis of ULMs from HLRCC (n = 16; FH-mutation confirmed) and non-syndromic (NS) patients (n = 12) identified a significantly higher protein:transcript correlation in HLRCC (R = 0.35) vs. NS ULMs (R = 0.242, MWU p = 0.0015). Co-altered proteins and transcripts (228) included antioxidant response element (ARE) target genes, such as thioredoxin reductase 1 (TXNRD1), and correlated with activation of NRF2-mediated oxidative stress response signaling in HLRCC ULMs. We confirm 185 transcripts previously described as altered between HLRCC and NS ULMs, 51 co-altered at the protein level and several elevated in HLRCC ULMs are involved in regulating cellular metabolism and glycolysis signaling. Furthermore, 367 S-(2-succino)cysteine peptides were identified in HLRCC ULMs, of which sixty were significantly elevated in HLRCC vs. NS ULMs (LogFC = 1.86, MWU p < 0.0001). These results confirm and define novel proteogenomic alterations in uterine leiomyoma tissues collected from HLRCC patients and underscore conserved molecular alterations correlating with inactivation of the FH tumor suppressor gene.

Transcriptomic Analysis of Mouse Brain After Traumatic Brain Injury Reveals That the Angiotensin Receptor Blocker Candesartan Acts Through Novel Pathways.

Traumatic brain injury (TBI) results in complex pathological reactions, where the initial lesion is followed by secondary inflammation and edema. Our laboratory and others have reported that angiotensin receptor blockers (ARBs) have efficacy in improving recovery from traumatic brain injury in mice. Treatment of mice with a subhypotensive dose of the ARB candesartan results in improved functional recovery, and reduced pathology (lesion volume, inflammation and gliosis). In order to gain a better understanding of the molecular mechanisms through which candesartan improves recovery after controlled cortical impact injury (CCI), we performed transcriptomic profiling on brain regions after injury and drug treatment. We examined RNA expression in the ipsilateral hippocampus, thalamus and hypothalamus at 3 or 29 days post injury (dpi) treated with either candesartan (0.1 mg/kg) or vehicle. RNA was isolated and analyzed by bulk mRNA-seq. Gene expression in injured and/or candesartan treated brain region was compared to that in sham vehicle treated mice in the same brain region to identify genes that were differentially expressed (DEGs) between groups. The most DEGs were expressed in the hippocampus at 3 dpi, and the number of DEGs reduced with distance and time from the lesion. Among pathways that were differentially expressed at 3 dpi after CCI, candesartan treatment altered genes involved in angiogenesis, interferon signaling, extracellular matrix regulation including integrins and chromosome maintenance and DNA replication. At 29 dpi, candesartan treatment reduced the expression of genes involved in the inflammatory response. Some changes in gene expression were confirmed in a separate cohort of animals by qPCR. Fewer DEGs were found in the thalamus, and only one in the hypothalamus at 3 dpi. Additionally, in the hippocampi of sham injured mice, 3 days of candesartan treatment led to the differential expression of 384 genes showing that candesartan in the absence of injury had a powerful impact on gene expression specifically in the hippocampus. Our results suggest that candesartan has broad actions in the brain after injury and affects different processes at acute and chronic times after injury. These data should assist in elucidating the beneficial effect of candesartan on recovery from TBI.

FOXP3 protects conventional human T cells from premature restimulation-induced cell death.

The adaptive immune response relies on specific apoptotic programs to maintain homeostasis. Conventional effector T cell (Tcon) expansion is constrained by both forkhead box P3 (FOXP3)+-regulatory T cells (Tregs) and restimulation-induced cell death (RICD), a propriocidal apoptosis pathway triggered by repeated stimulation through the T-cell receptor (TCR). Constitutive FOXP3 expression protects Tregs from RICD by suppressing SLAM-associated protein (SAP), a key adaptor protein that amplifies TCR signaling strength. The role of transient FOXP3 induction in activated human CD4 and CD8 Tcons remains unresolved, but its expression is inversely correlated with acquired RICD sensitivity. Here, we describe a novel role for FOXP3 in protecting human Tcons from premature RICD during expansion. Unlike FOXP3-mediated protection from RICD in Tregs, FOXP3 protects Tcons through a distinct mechanism requiring de novo transcription that does not require SAP suppression. Transcriptome profiling and functional analyses of expanding Tcons revealed that FOXP3 enhances expression of the SLAM family receptor CD48, which in turn sustains basal autophagy and suppresses pro-apoptotic p53 signaling. Both CD48 and FOXP3 expression reduced p53 accumulation upon TCR restimulation. Furthermore, silencing FOXP3 expression or blocking CD48 decreased the mitochondrial membrane potential in expanding Tcons with a concomitant reduction in basal autophagy. Our findings suggest that FOXP3 governs a distinct transcriptional program in early-stage effector Tcons that maintains RICD resistance via CD48-dependent protective autophagy and p53 suppression.

MutEnricher: a flexible toolset for somatic mutation enrichment analysis of tumor whole genomes.

BACKGROUND: Analysis of somatic mutations from tumor whole exomes has fueled discovery of novel cancer driver genes. However, ~ 98% of the genome is non-coding and includes regulatory elements whose normal cellular functions can be disrupted by mutation. Whole genome sequencing (WGS), on the other hand, allows for identification of non-coding somatic variation and expanded estimation of background mutation rates, yet fewer computational tools exist for specific interrogation of this space. RESULTS: We present MutEnricher, a flexible toolset for investigating somatic mutation enrichment in both coding and non-coding genomic regions from WGS data. MutEnricher contains two distinct modules for these purposes that provide customizable options for calculating sample- and feature-specific background mutation rates. Additionally, both MutEnricher modules calculate feature-level and local, or "hotspot," somatic mutation enrichment statistics. CONCLUSIONS: MutEnricher is a flexible software package for investigating somatic mutation enrichment that is implemented in Python, is freely available, can be efficiently parallelized, and is highly configurable to researcher's specific needs. MutEnricher is available online at https://github.com/asoltis/MutEnricher .

Hemophilia A Inhibitor Subjects Show Unique PBMC Gene Expression Profiles That Include Up-Regulated Innate Immune Modulators.

Formation of pathological anti-FVIII antibodies, or "inhibitors," is the most serious complication of therapeutic FVIII infusions, affecting up to 1/3 of severe Hemophilia A (HA) patients. Inhibitor formation is a classical T-cell dependent adaptive immune response. As such, it requires help from the innate immune system. However, the roles of innate immune cells and mechanisms of inhibitor development vs. immune tolerance, achieved with or without Immune Tolerance Induction (ITI) therapy, are not well-understood. To address these questions, temporal transcriptomics profiling of FVIII-stimulated peripheral blood mononuclear cells (PBMCs) was carried out for HA subjects with and without a current or historic inhibitor using RNA-Seq. PBMCs were isolated from 40 subjects in the following groups: HA with an inhibitor that resolved either following ITI or spontaneously; HA with a current inhibitor; HA with no inhibitor history and non-HA controls. PBMCs were stimulated with 5 nM FVIII and RNA was isolated 4, 16, 24, and 48 h following stimulation. Time-series differential expression analysis was performed and distinct transcriptional signatures were identified for each group, providing clues as to cellular mechanisms leading to or accompanying their disparate anti-FVIII antibody responses. Subjects with a current inhibitor showed differential expression of 56 genes and a clustering analysis identified three major temporal profiles. Interestingly, gene ontology enrichments featured innate immune modulators, including NLRP3, TLR8, IL32, CLEC10A, and COLEC12. NLRP3 and TLR8 are associated with enhanced secretion of the pro-inflammatory cytokines IL-1ß and TNFα, while IL32, which has several isoforms, has been associated with both inflammatory and regulatory immune processes. RNA-Seq results were validated by RT-qPCR, ELISAs, multiplex cytokine analysis, and flow cytometry. The inflammatory status of HA patients suffering from an ongoing inhibitor includes up-regulated innate immune modulators, which may act as ongoing danger signals that influence the responses to, and eventual outcomes of, ITI therapy.