The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

Characteristic chest CT findings for progressive cavities in Mycobacterium avium complex pulmonary disease: a retrospective cohort study

BACKGROUND: Although cavities are an important finding in Mycobacterium avium complex pulmonary disease (MAC-PD), there is little information regarding the types of cavities that indicate disease progression. This study was performed to identify cavity characteristics that were associated with disease progression in patients with MAC-PD. METHODS: This retrospective cohort study included 97 patients presenting with MAC-PD with cavities between December 2006 and June 2016. We compared initial and final computed tomography (CT) findings, classified 52 and 45 patients in the progressive and non-progressive cavity groups, respectively, and examined the progression-related imaging features in initial CT images. A progressive cavity was defined by more than two-fold increase in internal diameter or emergence of a new cavity around the initial cavity. RESULTS: Patients in the progressive group were older (p < 0.001), had a lower body mass index (p = 0.043), and showed higher diabetes complication rates (p = 0.005). The initial CT in the progressive group showed a longer maximum internal diameter of the cavity (p < 0.001) and higher rates of cavities close to the chest wall (p < 0.001), multiple cavities (p = 0.023), consolidation around the cavity (p < 0.001), atelectasis (p = 0.011), and pleural thickening (p < 0.001). Multivariable logistic regression analysis revealed that the maximum internal diameter of the cavity (odds ratio [OR]: 1.11, 95% confidence interval [CI]: 1.02–1.21; p=0.012) and consolidation around the cavity (OR: 16.15, 95% CI: 4.05–64.46; p < 0.001) were significantly associated with progressive cavities. In cavities with a maximum internal diameter of ≥10 mm and simultaneous consolidation, the probability of progression was as high as 96.2%. The 10-year mortality rates in the progressive and non-progressive cavity groups were 46.7 and 9.8% (p < 0.001), respectively, while the 10-year respiratory failure rates were 28.1 and 0%, respectively (p < 0.001). CONCLUSIONS: Large cavity size and consolidation on CT showed strong relationships with disease progression, which led to respiratory failure and high mortality rate

Improvement of exertional dyspnea and breathing pattern of inspiration to expiration after bronchial thermoplasty

Abstract Background Bronchial thermoplasty (BT) is a bronchoscopic treatment that can ameliorate the symptoms of severe asthma. However, little is known about the mechanism by which BT improves exertional dyspnea without significantly changing the resting pulmonary function in asthmatics. To understand the mechanism, cardiopulmonary variables were investigated using cardiopulmonary exercise testing (CPET) in a patient with severe asthma before and after BT. Case presentation A 57-year-old Japanese man visited our hospital for consultation of the intractable asthma, which we managed with three treatment sessions of BT. Comparison of the findings pre-BT and at 1 year after BT demonstrated that (1) the resting tests for respiration showed no improvement in forced expiratory volume in 1 s, but the forced oscillation technique showed decreases in both inhalation and exhalation respiratory resistance values, and (2) the CPET results showed (i) improvement in exertional dyspnea, exercise endurance, and arterial oxygen saturation at the end of exercise; (ii) that the expiratory tidal volume exceeded the inspiratory tidal volume during exercise, which implied that a sufficient exhalation enabled longer inspiratory time and adequate oxygen absorption; and (iii) that an increase in respiratory frequency could be prevented throughout exercise. Conclusions This case report described a novel mechanism of BT in improving exertional dyspnea and exercise duration, which was brought about by ventilatory improvements related to the breathing pattern of inspiration to expiration

Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases.

Hybrid sensor kinase, which contains a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, conveys signals to its cognate response regulator by means of a His-Asp-His-Asp phosphorelay. We examined the multistep phosphorelay of a recombinant EvgAS system in Escherichia coli and performed in vitro quantitative analyses of phosphorylation by using Phos-tag SDS-PAGE. Replacement of Asp in the receiver domain of EvgS by Ala markedly promoted phosphorylation at His in the HK domain compared with that in wild-type EvgS. Similar Ala-substituted mutants of other hybrid sensor kinases BarA and ArcB showed similar characteristics. In the presence of sufficient ATP, autophosphorylation of the HK domain in the mutant progressed efficiently with nearly pseudo-first-order kinetics until the phosphorylation ratio reached a plateau value of more than 95% within 60 min, and the value was maintained until 180 min. However, both wild-type EvgS and the Ala-substituted mutant of His in the HPt domain showed a phosphorylation ratio of less than 25%, which gradually decreased after 10 min. These results showed that the phosphorylation level is regulated negatively by the receiver domain. Furthermore, our in vivo assays confirmed the existence of a similar hyperphosphorylation reaction in the HK domain of the EvgS mutant in which the Asp residue was replaced with Ala, confirming the validity of the control mechanism proposed from profiling of phosphorylation in vitro [corrected]

Autophosphorylation reactions of other hybrid sensor kinases, BarA and ArcB.

<p>(A) Phosphorylation sites in each domain of BarA and ArcB are shown. (B) Autophosphorylation reactions of wild-type BarA (WT) and the two mutants D718A and H861A were performed in the presence of 10 mM ATP, and the products were then analyzed by Phos-tag SDS-PAGE. The incubation times are shown above each lane. Each lane contained 2 μg of protein. Bands for each of the two site-specific phosphorylated forms were assigned and are shown by arrows on the right-hand side of the panel. (C) Autophosphorylation reactions of wild-type ArcB (WT) and the two mutants D576A and H717A were performed and analyzed in the same manner as BarA in B. (D) Values of the ratio of the phosphorylated forms to the total proteins in B and C were calculated by densitometry and are plotted versus the reaction times. Each plot was gained as average of three independent experiments using the same sample. Standard deviations were within almost 20%.</p

<i>In vivo</i> analysis of EvgS phosphorylation.

<p>(A) β-galactosidase assays to confirm the arabinose-induced activities of EvgS, EvgS1, and their mutants H721A, D1009A, and H1137A, expressed by a pBAD vector. When using <i>E</i>. <i>coli</i> MG1655 <i>evgS ydeP-lacZ</i> as a host cell, the activation of the EvgAS system induces transcription of <i>lacZ</i>, which controls the promoter of <i>ydeP</i> (evgA regulon). For both EvgS and EvgS1, the proteins without the Ala substitution at each phosphorylation site are described as WT. Data were represented by mean values and standard deviation bars obtained from three independent experiments. (B) <i>In vivo</i> profiling of autophosphorylation reactions of EvgS, EvgS1, and their mutants by using Phos-tag SDS-PAGE followed by Western blotting. Half of each lysate samples was boiled for 3 min to hydrolyze phosphoryl groups on His and Asp residues. Each lane contains 20 μg of the crude cellular protein. Bands for each of the two site-specific phosphorylated forms were assigned and are shown by arrows on the right-hand side of the panel.</p

Time-dependent changes in levels of phosphorylation of wild-type EvgS (WT) and the two mutants D1009A and H1137A.

<p>(A) Autophosphorylation reactions of WT, D1009A, and H1137A were performed in the presence of 30 mM ATP and analyzed by Phos-tag SDS-PAGE. The incubation times are shown above each lane. Each lane contained 2 μg of protein. (B) The values of the ratios of the phosphorylated forms to the total proteins were calculated by densitometry and are plotted versus the reaction times. (C) The values of the ratios for each phosphorylated form derived from the WT to the total proteins were calculated and are plotted versus the reaction times. Each plot was gained as average of three independent experiments using the same sample. Standard deviations were within almost 20%.</p

PCR primers used in this study.

<p>PCR primers used in this study.</p