Whole Blood RNA Research Articles

Improvement in Disease Activity in Refractory Juvenile Dermatomyositis Following Abatacept Therapy.

This open-label, 24-week study was conducted to evaluate the safety and efficacy of abatacept in patients with refractory juvenile dermatomyositis (DM). Ten patients ≥7 years of age with moderate disease activity were enrolled in a 24-week study to examine the safety of subcutaneous abatacept and patient responses to the treatment. The primary endpoint was the International Myositis Assessment and Clinical Studies (IMACS) group Definition Of Improvement (DOI). Secondary endpoints included safety, changes in the core set activity measures (CSMs) of the IMACS group and the Pediatric Rheumatology International Trials Organization, and improvements in disease activity based on the American College of Rheumatology (ACR)/EULAR response criteria for juvenile DM. Radiologists blinded with regard to participant data assessed magnetic resonance images (MRIs) of patient thigh muscles. Interferon (IFN)-regulated gene score was performed on whole-blood RNA samples using a NanoString assay, and cytokines were assessed using a Luminex assay. Five patients achieved DOI at week 12, and 9 patients achieved DOI at week 24, including 2 patients with minimal, 4 patients with moderate, and 3 patients with major improvement by the 2016 ACR/EULAR response criteria for juvenile DM when patients were assessed using the CSMs of the IMACS Group. Improvements from baseline were seen in all CSMs at weeks 12 and 24, except in muscle enzymes. Daily glucocorticoid doses decreased from a mean of 16.7 mg at baseline to 10.2 mg at week 24 (P=0.002). Average MRI muscle edema scores decreased from a mean baseline score of 5.3 to 2.3 at week 24 (P=0.01). Six patients had down-trending IFN-regulated gene scores and galectin-9 expression at week 24. Decreases in IFN-regulated gene scores and in levels of interferon-γ-inducible protein 10kDa, galectin-9, and interleukin-2 correlated with improvements in disease activity and in muscle edema shown on MRI. Eleven grade 2 or 3 treatment-emergent adverse events were observed. This open-label study demonstrated that abatacept may be beneficial for patients with treatment-refractory juvenile DM.

Diagnosis of Multisystem Inflammatory Syndrome in Children by a Whole-Blood Transcriptional Signature.

To identify a diagnostic blood transcriptomic signature that distinguishes multisystem inflammatory syndrome in children (MIS-C) from Kawasaki disease (KD), bacterial infections, and viral infections. Children presenting with MIS-C to participating hospitals in the United Kingdom and the European Union between April 2020 and April 2021 were prospectively recruited. Whole-blood RNA Sequencing was performed, contrasting the transcriptomes of children with MIS-C (n = 38) to those from children with KD (n = 136), definite bacterial (DB; n = 188) and viral infections (DV; n = 138). Genes significantly differentially expressed (SDE) between MIS-C and comparator groups were identified. Feature selection was used to identify genes that optimally distinguish MIS-C from other diseases, which were subsequently translated into RT-qPCR assays and evaluated in an independent validation set comprising MIS-C (n = 37), KD (n = 19), DB (n = 56), DV (n = 43), and COVID-19 (n = 39). In the discovery set, 5696 genes were SDE between MIS-C and combined comparator disease groups. Five genes were identified as potential MIS-C diagnostic biomarkers (HSPBAP1, VPS37C, TGFB1, MX2, and TRBV11-2), achieving an AUC of 96.8% (95% CI: 94.6%-98.9%) in the discovery set, and were translated into RT-qPCR assays. The RT-qPCR 5-gene signature achieved an AUC of 93.2% (95% CI: 88.3%-97.7%) in the independent validation set when distinguishing MIS-C from KD, DB, and DV. MIS-C can be distinguished from KD, DB, and DV groups using a 5-gene blood RNA expression signature. The small number of genes in the signature and good performance in both discovery and validation sets should enable the development of a diagnostic test for MIS-C.

Transcriptomic Approaches to Diagnosis of Inborn Errors of Immunity

Molecular diagnosis of inborn errors of immunity (IEI) leads to many benefits including more accurate prognosis, access to molecularly targeted therapies, and better informed genetic counseling for relatives and offspring. However, in practice, diagnostic testing is often limited to gene panels and clinical exome sequencing or rarely genome sequencing. As a result, treating physicians remain unable to identify a specific cause in the majority of suspected IEI patients using currently established technologies, even when a genetic cause is suspected based on similarly affected family members. Even once a diagnosis is made, selection of appropriate treatments provides additional challenges as response to immunomodulatory therapy is heterogeneous. Better strategies are needed to identify which groups of patients are most likely to benefit from different therapies. Determining the optimal diagnostic and therapeutic strategy requires a personalized approach bringing together expertise from multiple clinical specialties and basic scientists to apply new discoveries to patient care and improve outcomes for these complex patients. We hypothesize that adding transcriptomic profiling to existing genomic diagnostic approaches could help close these gaps, facilitating identification of the molecular diagnosis in a larger fraction of patients and monitoring the gene expression response to treatment. To test this, we plan to (1) deploy RNA sequencing of peripheral blood paired with DNA sequencing in a cohort of IEI patients with known (N = 10) IEIs representing a spectrum of disorders as classified by IUIS (e.g. X-linked SCID due to ILR2g, deficiency of adenosine deaminase 2, CD40L deficiency) to validate the pipeline for detecting known disorders, (2) apply the pipeline to a pilot cohort of participants with unknown disorders (N = 10) to assess diagnostic efficacy, and (3) perform longitudinal RNA sequencing in IEI patients undergoing treatment to understand predictors of treatment response. To date, we have obtained whole-blood total RNA from 5 participants (3 known, 2 unknown and pretreatment). In parallel, we have adapted the detection of RNA outliers pipeline (DROP) for local use to identify novel monogenic causes of IEI through detection of aberrant expression, aberrant splicing, and mono-allelic expression, and have tested the pipeline using publicly available, well-characterized datasets.

Nucleic acid biomarkers of immune response and cell and tissue damage in children with COVID-19 and MIS-C

Differential host responses in coronavirus disease 2019 (COVID-19) and multisystem inflammatory syndrome in children (MIS-C) remain poorly characterized. Here, we use next-generation sequencing to longitudinally analyze blood samples from pediatric patients with COVID-19 or MIS-C across three hospitals. Profiling of plasma cell-free nucleic acids uncovers distinct signatures of cell injury and death between COVID-19 and MIS-C, with increased multiorgan involvement in MIS-C encompassing diverse cell types, including endothelial and neuronal cells, and an enrichment of pyroptosis-related genes. Whole-blood RNA profiling reveals upregulation of similar pro-inflammatory pathways in COVID-19 and MIS-C but also MIS-C-specific downregulation of Tcell-associated pathways. Profiling of plasma cell-free RNA and whole-blood RNA in paired samples yields different but complementary signatures for each disease state. Our work provides a systems-level view of immune responses and tissue damage in COVID-19 and MIS-C and informs future development of new disease biomarkers.

Genetic and Molecular Distinctions Between Axial Psoriatic Arthritis and Radiographic Axial Spondyloarthritis: Post Hoc Analyses from Four Phase3 Clinical Trials.

Emerging evidence suggests psoriatic arthritis (PsA)with axial involvement (axPsA) and radiographic axial spondyloarthritis (r-axSpA) maypossibly represent distinct disorders, with some differing clinical manifestations, genetic associations, and radiographic findings. Moreover, axPsA and r-axSpA may respond differently to therapies: guselkumab (interleukin [IL]-23p19 subunit inhibitor [i]) and ustekinumab (IL-12/23p40i) demonstrated improvements in axial symptoms in patients with PsA; however, neither risankizumab (IL-23p19i) nor ustekinumab demonstrated efficacy versus placebo in patients with r-axSpA. Current analyses aim to further understand potential molecular distinctions between axPsA and r-axSpA and examine the pharmacodynamic effects of guselkumab in patients with axPsA and those with PsA without axial involvement (non-axPsA). Posthoc analyses utilized biomarker data from blood and serum samples collected from a subset of participants in phase3 studies of ustekinumab in r-axSpA and guselkumab in PsA (DISCOVER-1 and DISCOVER-2). Participants with axPsA were identified by investigator-verified sacroiliitis (imaging-confirmed)and axial symptoms. HLA mapping, serum cytokine analysis, and whole-blood RNA sequencing were conducted. Relative to r-axSpA, patients with axPsA had a lower prevalence of HLA-B27, HLA-C01, and HLA-C02 alleles and a higher prevalence of HLA-B13, HLA-B38, HLA-B57, HLA-C06, and HLA-C12 alleles. Compared with r-axSpA, patients with axPsA had elevated baseline levels of serum IL-17A and IL-17F cytokines, enrichment of IL-17 and IL-10 pathway-associated genes, and neutrophil gene markers. Across axPsA and non-axPsA cohorts, reductions in cytokine levels and normalization ofpathway-associated gene expressionwith guselkumab treatment were comparable. The differences in HLA genetic associations, serum cytokines, and enrichment scores support the concept that axPsA and r-axSpA may be distinct disorders. The comparable pharmacodynamic effects of guselkumab on cytokine levels and pathway-associated genes observed in patients with axPsA and non-axPsA are consistent with demonstrated clinical improvements across PsA cohorts. These findings contribute to the understanding of potential genetic and molecular distinctions between axPsA and r-axSpA. ClinicalTrials.gov identifiers, NCT03162796, NCT0315828, NCT02437162, and NCT02438787.

Development of a Novel Clinicomolecular Risk Index to Enhance Mortality Prediction and Immunological Stratification of Adults Hospitalized with Sepsis in Sub-Saharan Africa: A Pilot Study from Uganda.

The global burden of sepsis is concentrated in sub-Saharan Africa (SSA), where epidemic HIV and unique pathogen diversity challenge the effective management of severe infections. In this context, patient stratification based on biomarkers of a dysregulated host response may identify subgroups more likely to respond to targeted immunomodulatory therapeutics. In a prospective cohort of adults hospitalized with suspected sepsis in Uganda, we applied machine learning methods to develop a prediction model for 30-day mortality that integrates physiology-based risk scores with soluble biomarkers reflective of key domains of sepsis immunopathology. After model evaluation and internal validation, whole-blood RNA sequencing data were analyzed to compare biological pathway enrichment and inferred immune cell profiles between patients assigned differential model-based risks of mortality. Of 260 eligible adults (median age, 32 years; interquartile range, 26-43 years; 59.2% female, 53.9% living with HIV), 62 (23.8%) died by 30 days after hospital discharge. Among 14 biomarkers, soluble tumor necrosis factor receptor 1 (sTNFR1) and angiopoietin 2 (Ang-2) demonstrated the greatest importance for mortality prediction in machine learning models. A clinicomolecular model integrating sTNFR1 and Ang-2 with the Universal Vital Assessment (UVA) risk score optimized 30-day mortality prediction across multiple performance metrics. Patients assigned to the high-risk, UVA-based clinicomolecular subgroup exhibited a transcriptional profile defined by proinflammatory innate immune and necroptotic pathway activation, T-cell exhaustion, and expansion of key immune cell subsets including regulatory and gamma-delta T cells. Clinicomolecular stratification of adults with suspected sepsis in Uganda enhanced 30-day mortality prediction and identified a high-risk subgroup with a therapeutically targetable immunological profile. Further studies are needed to advance pathobiologically informed sepsis management in SSA.

Blood RNA alternative splicing events as diagnostic biomarkers for infectious disease

Assays detecting blood transcriptome changes are studied for infectious disease diagnosis. Blood-based RNA alternative splicing (AS) events, which have not been well characterized in pathogen infection, have potential normalization and assay platform stability advantages over gene expression for diagnosis. Here, we present a computational framework for developing AS diagnostic biomarkers. Leveraging a large prospective cohort of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and whole-blood RNA sequencing (RNA-seq) data, we identify a major functional AS program switch upon viral infection. Using an independent cohort, we demonstrate the improved accuracy of AS biomarkers for SARS-CoV-2 diagnosis compared with six reported transcriptome signatures. We then optimize a subset of AS-based biomarkers to develop microfluidic PCR diagnostic assays. This assay achieves nearly perfect test accuracy (61/62= 98.4%) using a naive principal component classifier, significantly more accurate than a gene expression PCR assay in the same cohort. Therefore, our RNA splicing computational framework enables a promising avenue for host-response diagnosis of infection.

Biallelic variants in the calpain regulatory subunit CAPNS1 cause pulmonary arterial hypertension

PurposeThe aim of this study was to identify the monogenic cause of pulmonary arterial hypertension (PAH), a multifactorial and often fatal disease, in 2 unrelated consanguine families. MethodsWe performed exome sequencing and validated variant pathogenicity by whole-blood RNA and protein expression analysis in both families. Further RNA sequencing of preserved lung tissue was performed to investigate the consequences on selected genes that are involved in angiogenesis, proliferation, and apoptosis. ResultsWe identified 2 rare biallelic variants in CAPNS1, encoding the regulatory subunit of calpain. The variants cosegregated with PAH in the families. Both variants lead to loss of function (LoF), which is demonstrated by aberrant splicing resulting in the complete absence of the CAPNS1 protein in affected patients. No other LoF CAPNS1 variant was identified in the genome data of more than 1000 patients with unresolved PAH. ConclusionThe calpain holoenzyme was previously linked to pulmonary vascular development and progression of PAH in patients. We demonstrated that biallelic LoF variants in CAPNS1 can cause idiopathic PAH by the complete absence of CAPNS1 protein. Screening of this gene in patients who are affected by PAH, especially with suspected autosomal recessive inheritance, should be considered.

Associations between circulating interferon and kynurenine/tryptophan pathway metabolites: support for a novel potential mechanism for cognitive dysfunction in SLE

ObjectiveQuinolinic acid (QA), a kynurenine (KYN)/tryptophan (TRP) pathway metabolite, is an N-methyl-D-aspartate receptor agonist that can produce excitotoxic neuron damage. Type I and II interferons (IFNs) stimulate the KYN/TRP pathway,...

PSIX-16 Urine Metabolome and Whole Blood Transcriptome of Beef Steers with low or High Residual Feed Intake

Abstract We analyzed the metabolome of urine samples to identify urinary metabolic biomarkers and whole blood transcriptome-based gene set enrichment to identify key pathways associated with divergent selection for low or high RFI in beef cattle. Low-RFI beef steers (n = 8; RFI = - 1.93 kg/d) and high-RFI beef steers (n = 8; RFI = + 2.01kg/d) were selected from a group of 56 growing crossbred beef steers after a 49-d performance testing period. At the end of the 49-d period, weekly urine and blood samples were collected three times from the low- and high-RFI steers. Metabolome analysis of the urine samples was conducted using a liquid chromatography–mass spectrometer and biomarker analysis of the metabolome data was performed to identify candidate biomarkers (FDR ≤ 0.05; AUC ≥ 0.85) associated with RFI. Whole-blood RNA sequencing was performed on an Illumina sequencer and gene set enrichment analysis (GSEA) was used to analyze differentially expressed (FDR ≤ 0.05) pathways. A total number of 557 metabolites were detected and identified. Biomarker analysis of the metabolome data identified N-acetyl-L-tyrosine, O-methyl-L-threonine, uridine, and threoninyl-hydroxyproline as candidate biomarkers (FDR ≤ 0.05; AUC > 0.85) of RFI. Results of GSEA revealed pathways associated with protein metabolism, cellular responses to external stimuli, and stress were differentially inhibited in high-RFI compared with low-RFI beef cattle, while pathways associated with binding and uptake of ligands by scavenger receptors and erythrocytes release/take up oxygen were differentially enriched (FDR < 0.05) in high-RFI beef steers. Taken together, our results revealed that urine is a potential source of metabolite biomarkers associated with RFI and beef steers divergently selected for low or high RFI have differential expressions of genes related to protein metabolism and stress responsiveness. high RFI revealed differential expressions of genes related to protein metabolism and stress responsiveness.

The Effect of Tropical Temperatures on the Quality of RNA Extracted from Stabilized Whole-Blood Samples.

Whole-blood-derived transcriptional profiling is widely used in biomarker discovery, immunological research, and therapeutic development. Traditional molecular and high-throughput transcriptomic platforms, including molecular assays with quantitative PCR (qPCR) and RNA-sequencing (RNA-seq), are dependent upon high-quality and intact RNA. However, collecting high-quality RNA from field studies in remote tropical locations can be challenging due to resource restrictions and logistics of post-collection processing. The current study tested the relative performance of the two most widely used whole-blood RNA collection systems, PAXgene® and Tempus™, in optimal laboratory conditions as well as suboptimal conditions in tropical field sites, including the effects of extended storage times and high storage temperatures. We found that Tempus™ tubes maintained a slightly higher RNA quantity and integrity relative to PAXgene® tubes at suboptimal tropical conditions. Both PAXgene® and Tempus™ tubes gave similar RNA purity (A260/A280). Additionally, Tempus™ tubes preferentially maintained the stability of mRNA transcripts for two reference genes tested, Succinate dehydrogenase complex, subunit A (SDHA) and TATA-box-binding protein (TBP), even when RNA quality decreased with storage length and temperature. Both tube types preserved the rRNA transcript 18S ribosomal RNA (18S) equally. Our results suggest that Tempus™ blood RNA collection tubes are preferable to PAXgene® for whole-blood collection in suboptimal tropical conditions for RNA-based studies in resource-limited settings.

Rumen-protected methionine during heat stress alters mTOR, insulin signaling, and 1-carbon metabolism protein abundance in liver, and whole-blood transsulfuration pathway genes in Holstein cows

We investigated effects of rumen-protected Met (RPM) during a heat stress (HS) challenge on (1) hepatic abundance of mTOR, insulin, and antioxidant signaling proteins, (2) enzymes in 1-carbon metabolism, and (3) innate immunity. Holstein cows (n = 32; mean ± standard deviation, 184 ± 59 d in milk) were randomly assigned to 1 of 2 environmental groups, and 1 of 2 diets [total mixed ration (TMR) with RPM (Smartamine M; 0.105% dry matter as top-dress) or TMR without (CON); n = 16/diet] in a split-plot crossover design. There were 2 periods with 2 phases. During phase 1 (9 d), all cows were in thermoneutral conditions (TN; temperature-humidity index = 60 ± 3) and fed ad libitum. During phase 2 (9 d), half the cows (n = 8/diet) were exposed to HS using electric heat blankets. The other half (n = 8/diet) remained in TN, but was pair-fed to HS counterparts. After a 14-d washout and 7-d adaptation period, the study was repeated (period 2) and environmental treatments were inverted relative to phase 2, but dietary treatments were the same. Blood was collected on d 6 of each phase 2 to measure immune function and isolate whole-blood RNA. Liver biopsies were performed at the end of each period for cystathione β-synthase (CBS) and methionine adenosyltransferase activity, glutathione concentration, and protein abundance. Data were analyzed using PROC MIXED in SAS. Abundance of CUL3, inhibitor of antioxidant responses, tended to be downregulated by HS suggesting increased oxidative stress. Heat-shock protein 70 abundance was upregulated by HS. Phosphorylated mTOR abundance was greater overall with RPM, suggesting an increase in pathway activity. An environment × diet (E × D) effect was observed for protein kinase B (AKT), whereas there was a tendency for an interaction for phosphorylated AKT. Abundance of AKT was upregulated in CON cows during HS versus TN, this was not observed in RPM cows. For phosphorylated AKT, tissue from HS cows fed CON had greater abundance compared with all other treatments. The same effect was observed for EIF2A (translation initiation) and SLC2A4 (insulin-induced glucose uptake). An E × D effect was observed for INSR due to upregulation in CON cows during HS versus TN cows fed CON or RPM. There was an E × D effect for CBS, with lower activity in RPM versus CON cows during HS. The CON cows tended to have greater CBS during HS versus TN. An E × D effect was observed for methionine adenosyltransferase, with lower activity in RPM versus CON during HS. Although activity increased in CON during HS versus TN, RPM cows tended to have greater activity during TN. Neutrophil and monocyte oxidative burst and monocyte phagocytosis decreased with HS. An (E × D) effect was observed for whole-blood mRNA abundance of CBS, SOD1 and CSAD; RPM led to upregulation during TN versus HS. Regardless of diet, CDO1, CTH, and SOD1 decreased with HS. Although HS increased hepatic HSP70 and seemed to alter antioxidant signaling, feeding RPM may help cows maintain homeostasis in mTOR, insulin signaling, and 1-carbon metabolism. Feeding RPM also may help maintain whole-blood antioxidant response during HS, which is an important aspect of innate immune function.

Spontaneous NLRP3 inflammasome-driven IL-1-β secretion is induced in severe COVID-19 patients and responds to anakinra treatment

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may result in a severe pneumonia associated with elevation of blood inflammatory parameters, reminiscent of cytokine storm syndrome. Steroidal anti-inflammatory therapies have shown efficacy in reducing mortality in critically ill patients; however, the mechanisms by which SARS-CoV-2 triggers such an extensive inflammation remain unexplained.ObjectivesTo dissect the mechanisms underlying SARS-CoV-2–associated inflammation in patients with severe coronavirus disease 2019 (COVID-19), we studied the role of IL-1β, a pivotal cytokine driving inflammatory phenotypes, whose maturation and secretion are regulated by inflammasomes.MethodsWe analyzed nod-like receptor protein 3 pathway activation by means of confocal microscopy, plasma cytokine measurement, cytokine secretion following in vitro stimulation of blood circulating monocytes, and whole-blood RNA sequencing. The role of open reading frame 3a SARS-CoV-2 protein was assessed by confocal microscopy analysis following nucleofection of a monocytic cell line.ResultsWe found that circulating monocytes from patients with COVID-19 display ASC (adaptor molecule apoptotic speck like protein–containing a CARD) specks that colocalize with nod-like receptor protein 3 inflammasome and spontaneously secrete IL-1β in vitro. This spontaneous activation reverts following patient’s treatment with the IL-1 receptor antagonist anakinra. Transfection of a monocytic cell line with cDNA coding for the ORF3a SARS-CoV-2 protein resulted in ASC speck formation.ConclusionsThese results provide further evidence that IL-1β targeting could represent an effective strategy in this disease and suggest a mechanistic explanation for the strong inflammatory manifestations associated with COVID-19.

Monocytic Subsets Impact Cerebral Cortex and Cognition: Differences Between Healthy Subjects and Patients With First-Episode Schizophrenia.

Monocytes are a highly heterogeneous population subcategorized into classical, intermediate and nonclassical subsets. How monocytes and their subsets may shape brain structures and functions in schizophrenia remains unclear. The primary goal of this cross-sectional study was to investigate monocytic subsets and their specific signature genes in regulation of cerebral cortical thickness and cognitive functions in first-episode schizophrenia (FES) patients. Whole-blood RNA sequencing of 128 FES patients and 111 healthy controls (HCs) were conducted and monocyte-specific differentially expressed genes were further analyzed. The MATRICS Consensus Cognitive Battery (MCCB) test, cortical neuroimaging and flow cytometric staining of peripheral blood monocytic subsets were performed among the participants. Significant changes in expressions of 54 monocytic signature genes were found in patients, especially for intermediate and nonclassical monocytic subsets with the most outstanding alterations being downregulated S100 Calcium Binding Protein A (S100A) and upregulated Interferon Induced Transmembrane Protein (IFITM) family members, respectively. Meanwhile, percentage of blood nonclassical monocytes was decreased in patients. Cortical thicknesses and MCCB performance were expectantly reduced and weaker intra-relationships among monocytic signature genes and cortices, respectively, were noted in patients compared to HCs. Monocytic genes were negatively associated with both cortical thicknesses and cognition in HCs, which was interestingly weakened or even reversed in patients, with nonclassical monocytic genes showing the greatest statistical significance. This study reveals that while monocytes may have negative effects on brain structure and cognition, the ameliorated phenomenon observed in schizophrenia may reflect an (mal)adaptive change of monocytes at early stage of the disorder.

RNASE6 is a novel modifier of APOE-ε4 effects on cognition

Apolipoprotein E4 (APOE-ε4), the strongest common genetic risk factor for Alzheimer's disease (AD), contributes to worse cognition in older adults. However, many APOE-ε4 carriers remain cognitively normal throughout life, suggesting that neuroprotective factors may be present in these individuals. In this study, we leverage whole-blood RNA sequencing (RNAseq) from 324 older adults to identify genetic modifiers of APOE-ε4 effects on cognition. Expression of RNASE6 interacted with APOE-ε4 status (p = 4.35 × 10−8) whereby higher RNASE6 expression was associated with worse memory at baseline among APOE-ε4 carriers. This interaction was replicated using RNAseq data from the prefrontal cortex in an independent dataset (N = 535; p = 0.002), suggesting the peripheral effect of RNASE6 is also present in brain tissue. RNASE6 encodes an antimicrobial peptide involved in innate immune response and has been previously observed in a gene co-expression network module with other AD-related inflammatory genes, including TREM2 and MS4A. Together, these data implicate neuroinflammation in cognitive decline, and suggest that innate immune signaling may be detectable in blood and confer differential susceptibility to AD depending on APOE-ε4.

A preliminary, observational study using whole-blood RNA sequencing reveals differential expression of inflammatory and bone markers post-implantation of percutaneous osseointegrated prostheses.

AimsWhile the benefits of direct skeletal attachment of artificial limbs are well recognized, device failure due to infection and insufficient osseointegration remain obstacles to obtaining consistently successful outcomes. Currently, the potential for device failure is assessed by subjective pain, clinical function scores, radiographic evidence of bone atrophy, and the presence of radiolucent lines at the bone-implant interface, and subjective pain and function scores. Our hypothesis is that measurable biological indices might add another objective means to assess trends toward bone and stomal healing. This longitudinal cohort study was undertaken to identify potential serological biomarkers suggestive of bone remodeling and the presence of stomal tissue inflammation.MethodsTen unilateral transfemoral amputee veterans, who were implanted with a percutaneous osseointegrated (OI) skeletal limb docking system, were recruited to participate in this IRB-approved study. Venous blood samples were obtained from before the Stage 1 Surgery up to 1 year following the Stage 2 Surgery. Whole-blood RNA was extracted, sequenced, mapped, and analyzed. Of the significant differentially expressed (DEGs) genes (p<0.05) identified, four genes of interest (IL12B, IL33, COL2A1, and SOST) were validated using qPCR. Enrichment analysis was performed to identify significant (p<0.01) Gene Ontology (GO) terms.ResultsMost differentially expressed genes were only detected at PoS1 immediately after the first surgery. Of the significant genes identified, IL12B and IL33 were related to inflammation, and COL2A1 and SOST were associated with bone remodeling. These four genes were identified with greater than 20 log fold-change.ConclusionWhole-blood RNA-seq data from 10 patients who previously underwent percutaneous osseointegrated lower limb implantation revealed four genes of interest that are known to be involved in inflammation or bone remodeling. If verified in future studies, these genes may serve as markers for predicting optimal bone remodeling and stomal tissue healing following OI device implantation.

A Pilot Study of Whole-Blood Transcriptomic Analysis to Identify Genes Associated with Repetitive Low-Level Blast Exposure in Career Breachers.

Repetitive low-level blast exposure is one of the major occupational health concerns among US military service members and law enforcement. This study seeks to identify gene expression using microRNA and RNA sequencing in whole-blood samples from experienced breachers and unexposed controls. We performed experimental RNA sequencing using Illumina’s HiSeq 2500 Sequencing System, and microRNA analysis using NanoString Technology nCounter miRNA expression panel in whole-blood total RNA samples from 15 experienced breachers and 14 age-, sex-, and race-matched unexposed controls. We identified 10 significantly dysregulated genes between experienced breachers and unexposed controls, with FDR corrected <0.05: One upregulated gene, LINC00996 (long intergenic non-protein coding RNA 996); and nine downregulated genes, IGLV3-16 (immunoglobulin lambda variable 3-16), CD200 (CD200 molecule), LILRB5 (leukocyte immunoglobulin-like receptor B5), ZNF667-AS1 (ZNF667 antisense RNA 1), LMOD1 (leiomodin 1), CNTNAP2 (contactin-associated protein 2), EVPL (envoplakin), DPF3 (double PHD fingers 3), and IGHV4-34 (immunoglobulin heavy variable 4-34). The dysregulated gene expressions reported here have been associated with chronic inflammation and immune response, suggesting that these pathways may relate to the risk of lasting neurological symptoms following high exposures to blast over a career.

TGF-β1 is associated with deficits in cognition and cerebral cortical thickness in first-episode schizophrenia.

Background:Evidence indicates that cytokines are associated with cognitive deficits in schizophrenia; however, the underlying brain–behaviour mechanisms remain unclear. We hypothesized that aberrations in brain structural connectivity mediate the cytokine effect in schizophrenia.Methods:In this study, we recruited patients with first-episode schizophrenia (n = 75, average illness duration 12.3 months, average medication period 0.6 days) and healthy controls (n = 44) of both sexes. We first conducted whole-blood RNA sequencing to detect differentially expressed genes. We also explored transcriptomic data on the dorsal lateral prefrontal cortices (dlPFC) retrieved from the CommonMind Consortium for gene functional clustering; we measured plasma transforming growth factor β1 (TGF-β1) levels by enzyme-linked immunosorbent assay; we acquired high-resolution T1-weighted MRI data on cortical thickness MRI; and we assessed cognitive function using the validated Chinese version of the MATRICS Consensus Cognitive Battery. We compared these parameters in patients with schizophrenia and controls, and analyzed their associations.Results:Patients with schizophrenia had higher TGF-β1 at both the mRNA level (log2 fold change = 0.24; adjusted p = 0.026) and the protein level (12.85 ± 6.01 μg/mL v. 8.46 ± 5.15 μg/mL, adjusted p < 0.001) compared to controls. Genes coexpressed with TGFB1 in the dlPFC were less abundant in patients with schizophrenia compared to healthy controls. In patients with schizophrenia, TGF-β1 protein levels were inversely correlated with cortical thickness, especially of the lateral occipital cortex (r = −0.47, adjusted p = 0.001), and with the MATRICS Consensus Cognitive Battery visual learning and memory domain (r = −0.50, adjusted p < 0.001). We found a complete mediation effect of the thickness of the lateral occipital cortex on the negative relationship between TGF-β1 and visual cognition (p < 0.05).Limitations:We did not explore the effect of other blood cytokines on neurocognitive performance and cortical thickness. Participants from the CommonMind Consortium did not all have first-episode schizophrenia and they were not all antipsychotic-naive, so we could not exclude an effect of antipsychotics on TGF-β1 signalling in the dlPFC. The sample size and cross-sectional design of our study were additional limitations.Conclusion:These findings highlighted an association between upregulated blood levels of TGF-β1 and impairments in brain structure and function in schizophrenia.

Quantitative Real-Time RT-PCR Systems to Detect SARS-CoV-2.

Since the outbreak of coronavirus disease 2019 (COVID-19) on the Diamond Princess cruise ship docked at Yokohama Port on February 3, 2020, real-time reverse transcription-polymerase chain reaction (RT-PCR) testing using nasopharyngeal swab samples from symptomatic and asymptomatic COVID-19 individuals has been the main way to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in almost all clinical laboratories in Japan. With the diffusion of sets containing the primers and probe, the gold standard real-time RT-PCR test has permeated throughout the country. To prevent the spread of infection, real-time RT-PCR testing is important to confirm whether people are positive, asymptomatic, or negative for COVID-19. Now, in addition to pharyngeal swab, saliva and blood samples can be used to detect SARS-CoV-2 RNA. Here, we introduce a clinical laboratory test performed using the High Pure viral nucleic acid kit and subsequent real-time RT-PCR system to detect SARS-CoV-2 RNA in serum, plasma, or whole blood in a hospital in Yokohama, Japan.

Development of a Blood-based Transcriptional Risk Score for Chronic Obstructive Pulmonary Disease.

Rationale: The ability of peripheral blood biomarkers to assess chronic obstructive pulmonary disease (COPD) risk and progression is unknown. Genetics and gene expression may capture important aspects of COPD-related biology that predict disease activity. Objectives: Develop a transcriptional risk score (TRS) for COPD and assess the contribution of the TRS and a polygenic risk score (PRS) for disease susceptibility and progression. Methods: We randomly split 2,569 COPDGene (Genetic Epidemiology of COPD) participants with whole-blood RNA sequencing into training (n = 1,945) and testing (n = 624) samples and used 468 ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points) COPD cases with microarray data for replication. We developed a TRS using penalized regression (least absolute shrinkage and selection operator) to model FEV1/FVC and studied the predictive value of TRS for COPD (Global Initiative for Chronic Obstructive Lung Disease 2-4), prospective FEV1 change (ml/yr), and additional COPD-related traits. We adjusted for potential confounders, including age and smoking. We evaluated the predictive performance of the TRS in the context of a previously derived PRS and clinical factors. Measurements and Main Results: The TRS included 147 transcripts and was associated with COPD (odds ratio, 3.3; 95% confidence interval [CI], 2.4-4.5; P < 0.001), FEV1 change (β, -17 ml/yr; 95% CI, -28 to -6.6; P = 0.002), and other COPD-related traits. In ECLIPSE cases, we replicated the association with FEV1 change (β, -8.2; 95% CI, -15 to -1; P = 0.025) and the majority of other COPD-related traits. Models including PRS, TRS, and clinical factors were more predictive of COPD (area under the receiver operator characteristic curve, 0.84) and annualized FEV1 change compared with models with one risk score or clinical factors alone. Conclusions: Blood transcriptomics can improve prediction of COPD and lung function decline when added to a PRS and clinical risk factors.