A global in vivo Drosophila RNAi screen identifies NOT3 as a conserved regulator of heart function.

Heart diseases are the most common causes of morbidity and death in humans. Using cardiac-specific RNAi-silencing in Drosophila, we knocked down 7061 evolutionarily conserved genes under conditions of stress. We present a first global roadmap of pathways potentially playing conserved roles in the cardiovascular system. One critical pathway identified was the CCR4-Not complex implicated in transcriptional and posttranscriptional regulatory mechanisms. Silencing of CCR4-Not components in adult Drosophila resulted in myofibrillar disarray and dilated cardiomyopathy. Heterozygous not3 knockout mice showed spontaneous impairment of cardiac contractility and increased susceptibility to heart failure. These heart defects were reversed via inhibition of HDACs, suggesting a mechanistic link to epigenetic chromatin remodeling. In humans, we show that a common NOT3 SNP correlates with altered cardiac QT intervals, a known cause of potentially lethal ventricular tachyarrhythmias. Thus, our functional genome-wide screen in Drosophila can identify candidates that directly translate into conserved mammalian genes involved in heart function.

Prognostic significance of chronic kidney disease and impaired renal function in Japanese patients with COVID-19.

BACKGROUND: Renal impairment is a predictor of coronavirus disease (COVID-19) severity. No studies have compared COVID-19 outcomes in patients with chronic kidney disease (CKD) and patients with impaired renal function without a prior diagnosis of CKD. This study aimed to identify the impact of pre-existing impaired renal function without CKD on COVID-19 outcomes. METHODS: This retrospective study included 3,637 patients with COVID-19 classified into three groups by CKD history and estimated glomerular filtration rate (eGFR) on referral: Group 1 (n = 2,460), normal renal function without a CKD history; Group 2 (n = 905), impaired renal function without a CKD history; and Group 3 (n = 272), history of CKD. We compared the clinical characteristics of these groups and assessed the effect of CKD and impaired renal function on critical outcomes (requirement for respiratory support with high-flow oxygen devices, invasive mechanical ventilation, or extracorporeal membrane oxygen, and death during hospitalization) using multivariable logistic regression. RESULTS: The prevalence of comorbidities (hypertension, diabetes, and cardiovascular disease) and incidence of inflammatory responses (white blood counts, and C-reactive protein, procalcitonin, and D-dimer levels) and complications (bacterial infection and heart failure) were higher in Groups 2 and 3 than that in Group 1. The incidence of critical outcomes was 10.8%, 17.7%, and 26.8% in Groups 1, 2, and 3, respectively. The mortality rate and the rate of requiring IMV support was lowest in Group 1 and highest in Group 3. Compared with Group 1, the risk of critical outcomes was higher in Group 2 (adjusted odds ratio [aOR]: 1.32, 95% confidence interval [CI]: 1.03-1.70, P = 0.030) and Group 3 (aOR: 1.94, 95% CI: 1.36-2.78, P < 0.001). Additionally, the eGFR was significantly associated with critical outcomes in Groups 2 (odds ratio [OR]: 2.89, 95% CI: 1.64-4.98, P < 0.001) and 3 (OR: 1.87, 95% CI: 1.08-3.23, P = 0.025) only. CONCLUSIONS: Clinicians should consider pre-existing CKD and impaired renal function at the time of COVID-19 diagnosis for the management of COVID-19.

Clinical utilization of artificial intelligence-based COVID-19 pneumonia quantification using chest computed tomography - a multicenter retrospective cohort study in Japan.

BACKGROUND: Computed tomography (CT) imaging and artificial intelligence (AI)-based analyses have aided in the diagnosis and prediction of the severity of COVID-19. However, the potential of AI-based CT quantification of pneumonia in assessing patients with COVID-19 has not yet been fully explored. This study aimed to investigate the potential of AI-based CT quantification of COVID-19 pneumonia to predict the critical outcomes and clinical characteristics of patients with residual lung lesions. METHODS: This retrospective cohort study included 1,200 hospitalized patients with COVID-19 from four hospitals. The incidence of critical outcomes (requiring the support of high-flow oxygen or invasive mechanical ventilation or death) and complications during hospitalization (bacterial infection, renal failure, heart failure, thromboembolism, and liver dysfunction) was compared between the groups of pneumonia with high/low-percentage lung lesions, based on AI-based CT quantification. Additionally, 198 patients underwent CT scans 3 months after admission to analyze prognostic factors for residual lung lesions. RESULTS: The pneumonia group with a high percentage of lung lesions (N = 400) had a higher incidence of critical outcomes and complications during hospitalization than the low percentage group (N = 800). Multivariable analysis demonstrated that AI-based CT quantification of pneumonia was independently associated with critical outcomes (adjusted odds ratio [aOR] 10.5, 95% confidence interval [CI] 5.59-19.7), as well as with oxygen requirement (aOR 6.35, 95% CI 4.60-8.76), IMV requirement (aOR 7.73, 95% CI 2.52-23.7), and mortality rate (aOR 6.46, 95% CI 1.87-22.3). Among patients with follow-up CT scans (N = 198), the multivariable analysis revealed that the pneumonia group with a high percentage of lung lesions on admission (aOR 4.74, 95% CI 2.36-9.52), older age (aOR 2.53, 95% CI 1.16-5.51), female sex (aOR 2.41, 95% CI 1.13-5.11), and medical history of hypertension (aOR 2.22, 95% CI 1.09-4.50) independently predicted persistent residual lung lesions. CONCLUSIONS: AI-based CT quantification of pneumonia provides valuable information beyond qualitative evaluation by physicians, enabling the prediction of critical outcomes and residual lung lesions in patients with COVID-19.

Association between ABO blood group/genotype and COVID-19 in a Japanese population.

An association between coronavirus disease 2019 (COVID-19) and the ABO blood group has been reported. However, such an association has not been studied in the Japanese population on a large scale. Little is known about the association between COVID-19 and ABO genotype. This study investigated the association between COVID-19 and ABO blood group/genotype in a large Japanese population. All Japanese patients diagnosed with COVID-19 were recruited through the Japan COVID-19 Task Force between February 2020 and October 2021. We conducted a retrospective cohort study involving 1790 Japanese COVID-19 patients whose DNA was used for a genome-wide association study. We compared the ABO blood group/genotype in a healthy population (n = 611, control) and COVID-19 patients and then analyzed their associations and clinical outcomes. Blood group A was significantly more prevalent (41.6% vs. 36.8%; P = 0.038), and group O was significantly less prevalent (26.2% vs. 30.8%; P = 0.028) in the COVID-19 group than in the control group. Moreover, genotype OO was significantly less common in the COVID-19 group. Furthermore, blood group AB was identified as an independent risk factor for most severe diseases compared with blood group O [aOR (95% CI) = 1.84 (1.00-3.37)]. In ABO genotype analysis, only genotype AB was an independent risk factor for most severe diseases compared with genotype OO. Blood group O is protective, whereas group A is associated with the risk of infection. Moreover, blood group AB is associated with the risk of the "most" severe disease.

Clinical significance of prediabetes, undiagnosed diabetes and diagnosed diabetes on critical outcomes in COVID-19: Integrative analysis from the Japan COVID-19 task force.

AIM: Diabetes mellitus (DM) is a known risk factor for severe coronavirus disease 2019 (COVID-19), but the clinical impact of undiagnosed diabetes and prediabetes in COVID-19 are unclear particularly in Japan. We clarify the difference in clinical characteristics, including age, sex, body mass index and co-morbidities, laboratory findings and critical outcomes, in a large Japanese COVID-19 cohort without diabetes, with prediabetes, undiagnosed diabetes and diagnosed diabetes, and to identify associated risk factors. MATERIALS AND METHODS: This multicentre, retrospective cohort study used the Japan COVID-19 Task Force database, which included data on 2430 hospitalized COVID-19 patients from over 70 hospitals from February 2020 to October 2021. The prevalence of prediabetes, undiagnosed diabetes and diagnosed diabetes were estimated based on HbA1c levels or a clinical diabetes history. Critical outcomes were defined as the use of high-flow oxygen, invasive positive-pressure ventilation or extracorporeal membrane oxygenation, or death during hospitalization. RESULTS: Prediabetes, undiagnosed diabetes and diagnosed diabetes were observed in 40.9%, 10.0% and 23.0%, respectively. Similar to diagnosed diabetes, prediabetes and undiagnosed diabetes were risk factors for critical COVID-19 outcomes (adjusted odds ratio [aOR] [95% CI]: 2.13 [1.31-3.48] and 4.00 [2.19-7.28], respectively). HbA1c was associated with COVID-19 severity in prediabetes patients (aOR [95% CI]: 11.2 [3.49-36.3]), but not other groups. CONCLUSIONS: We documented the clinical characteristics and outcomes of Japanese COVID-19 patients according to HbA1c levels or diabetes co-morbidity. As well as undiagnosed and diagnosed diabetes, physicians should be aware of prediabetes related to COVID-19 severity.

Killer cell immunoglobulin-like receptor three domains long cytoplasmic tail 1 gene *007 may modulate disease progression of human immunodeficiency virus-1 infection in the Japanese population.

One of the KIR allele, KIR3DL1*007, was associated with the progression to acquired immunodeficiency syndrome and not with the susceptibility to HIV-1 infection in the Japanese and Indian populations, implying that KIR3DL1*007-positive NK cells might eliminate HIV-infected cells less effectively than NK cells bearing the other KIR3DL1 alleles or KIR3DS1 alleles.

CD8 T Cells Show Protection against Highly Pathogenic Simian Immunodeficiency Virus (SIV) after Vaccination with SIV Gene-Expressing BCG Prime and Vaccinia Virus/Sendai Virus Vector Boosts.

Toward development of a dual vaccine for human immunodeficiency virus type 1 (HIV-1) and tuberculosis infections, we developed a urease-deficient bacillus Calmette-Guérin (BCG) strain Tokyo172 (BCGΔurease) to enhance its immunogenicity. BCGΔurease expressing a simian immunodeficiency virus (SIV) Gag induced BCG antigen-specific CD4+ and CD8+ T cells more efficiently and more Gag-specific CD8+ T cells. We evaluated its protective efficacy against SIV infection in cynomolgus monkeys of Asian origin, shown to be as susceptible to infection with SIVmac251 as Indian rhesus macaques. Priming with recombinant BCG (rBCG) expressing SIV genes was followed by a boost with SIV gene-expressing LC16m8Δ vaccinia virus and a second boost with SIV Env-expressing Sendai virus. Eight weeks after the second boost, monkeys were repeatedly challenged with a low dose of SIVmac251 intrarectally. Two animals out of 6 vaccinees were protected, whereas all 7 control animals were infected without any early viral controls. In one vaccinated animal, which had the most potent CD8+ T cells in an in vitro suppression activity (ISA) assay of SIVmac239 replication, plasma viremia was undetectable throughout the follow-up period. Protection was confirmed by the lack of anamnestic antibody responses and detectable cell-associated provirus in various organs. Another monkey with a high ISA acquired a small amount of SIV, but it later became suppressed below the detection limit. Moreover, the ISA score correlated with SIV acquisition. On the other hand, any parameter relating anti-Env antibody was not correlated with the protection.IMPORTANCE Because both AIDS and tuberculosis are serious health threats in middle/low-income countries, development of a dual vaccine against them would be highly beneficial. To approach the goal, here we first assessed a urease-deficient bacillus Calmette-Guérin (BCG) for improvement of immunogenicity against both Mycobacterium tuberculosis and SIV. Second, we demonstrated the usefulness of Asian-origin cynomolgus monkeys for development of a preclinical AIDS vaccine by direct comparison with Indian rhesus macaques as the only validated hosts that identically mirror the outcomes of clinical trials, since the availability of Indian rhesus macaques is limited in countries other than the United States. Finally, we report the protective effect of a vaccination regimen comprising BCG, the highly attenuated vaccinia virus LC16m8Δ strain, and nontransmissible Sendai virus as safe vectors expressing SIV genes using repeated mucosal challenge with highly pathogenic SIVmac251. Identification of CD8+ T cells as a protective immunity suggests a future direction of AIDS vaccine development.

Viral load of SARS-CoV-2 Omicron is not high despite its high infectivity.

Patients infected with the Omicron variant of severe acute respiratory syndrome coronavirus 2 has increased worldwide since the beginning of 2022 and the variant has spread more rapidly than the Delta variant, which spread in the summer of 2021. It is important to clarify the cause of the strong transmissibility of the Omicron variant to control its spread. In 694 patients with coronavirus disease 2019, the copy numbers of virus in nasopharyngeal swab-soaked samples and the viral genotypes were examined using quantitative polymerase chain reaction (PCR) and PCR-based melting curve analysis, respectively. Whole-genome sequencing was also performed to verify the viral genotyping data. There was no significant difference (p = 0.052) in the copy numbers between the Delta variant cases (median 1.5 × 105 copies/µl, n = 174) and Omicron variant cases (median 1.2 × 105 copies/µl, n = 328). During this study, Omicron BA.1 cases (median 1.1 ×105 copies/µl, n = 275) began to be replaced by BA.2 cases (median 2.3 × 105 copies/µl, n = 53), and there was no significant difference between the two groups (p = 0.33). Our results suggest that increased infectivity of the Omicron variant and its derivative BA.2 is not caused by higher viral loads but by other factors, such as increased affinity to cell receptors or immune escape.

Viral loads and profile of the patients infected with SARS-CoV-2 Delta, Alpha, or R.1 variants in Tokyo.

The rapid spread of the Delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became a serious concern worldwide in summer 2021. We examined the copy number and variant types of all SARS-CoV-2-positive patients who visited our hospital from February to August 2021 using polymerase chain reaction (PCR) tests. Whole genome sequencing was performed for some samples. The R.1 variant (B.1.1.316) was responsible for most infections in March, replacing the previous variant (B.1.1.214); the Alpha (B.1.1.7) variant caused most infections in April and May; and the Delta variant (B.1.617.2) was the most prevalent in July and August. There was no significant difference in the copy numbers among the previous variant cases (n = 29, median 3.0 × 104 copies/µl), R.1 variant cases (n = 28, 2.1 × 105 copies/µl), Alpha variant cases (n = 125, 4.1 × 105 copies/µl), and Delta variant cases (n = 106, 2.4 × 105 copies/µl). Patients with Delta variant infection were significantly younger than those infected with R.1 and the previous variants, possibly because many elderly individuals in Tokyo were vaccinated between May and August. There was no significant difference in mortality among the four groups. Our results suggest that the increased infectivity of Delta variant may be caused by factors other than the higher viral loads. Clarifying these factors is important to control the spread of Delta variant infection.

Impact of upper and lower respiratory symptoms on COVID-19 outcomes: a multicenter retrospective cohort study.

BACKGROUND: Respiratory symptoms are associated with coronavirus disease 2019 (COVID-19) outcomes. However, the impacts of upper and lower respiratory symptoms on COVID-19 outcomes in the same population have not been compared. The objective of this study was to characterize upper and lower respiratory symptoms and compare their impacts on outcomes of hospitalized COVID-19 patients. METHODS: This was a multicenter, retrospective cohort study; the database from the Japan COVID-19 Task Force was used. A total of 3314 COVID-19 patients were included in the study, and the data on respiratory symptoms were collected. The participants were classified according to their respiratory symptoms (Group 1: no respiratory symptoms, Group 2: only upper respiratory symptoms, Group 3: only lower respiratory symptoms, and Group 4: both upper and lower respiratory symptoms). The impacts of upper and lower respiratory symptoms on the clinical outcomes were compared. The primary outcome was the percentage of patients with poor clinical outcomes, including the need for oxygen supplementation via high-flow oxygen therapy, mechanical ventilation, and extracorporeal membrane oxygenation or death. RESULTS: Of the 3314 COVID-19 patients, 605, 1331, 1229, and 1149 were classified as Group 1, Group 2, Group 3, and Group 4, respectively. In univariate analysis, patients in Group 2 had the best clinical outcomes among all groups (odds ratio [OR]: 0.21, 95% confidence interval [CI]: 0.11-0.39), while patients in Group 3 had the worst outcomes (OR: 3.27, 95% CI: 2.43-4.40). Group 3 patients had the highest incidence of pneumonia, other complications due to secondary infections, and thrombosis during the clinical course. CONCLUSIONS: Upper and lower respiratory tract symptoms had vastly different impacts on the clinical outcomes of COVID-19.

Clinical clustering with prognostic implications in Japanese COVID-19 patients: report from Japan COVID-19 Task Force, a nation-wide consortium to investigate COVID-19 host genetics.

BACKGROUND: The clinical course of coronavirus disease (COVID-19) is diverse, and the usefulness of phenotyping in predicting the severity or prognosis of the disease has been demonstrated overseas. This study aimed to investigate clinically meaningful phenotypes in Japanese COVID-19 patients using cluster analysis. METHODS: From April 2020 to May 2021, data from inpatients aged ≥ 18 years diagnosed with COVID-19 and who agreed to participate in the study were collected. A total of 1322 Japanese patients were included. Hierarchical cluster analysis was performed using variables reported to be associated with COVID-19 severity or prognosis, namely, age, sex, obesity, smoking history, hypertension, diabetes mellitus, malignancy, chronic obstructive pulmonary disease, hyperuricemia, cardiovascular disease, chronic liver disease, and chronic kidney disease. RESULTS: Participants were divided into four clusters: Cluster 1, young healthy (n = 266, 20.1%); Cluster 2, middle-aged (n = 245, 18.5%); Cluster 3, middle-aged obese (n = 435, 32.9%); and Cluster 4, elderly (n = 376, 28.4%). In Clusters 3 and 4, sore throat, dysosmia, and dysgeusia tended to be less frequent, while shortness of breath was more frequent. Serum lactate dehydrogenase, ferritin, KL-6, D-dimer, and C-reactive protein levels tended to be higher in Clusters 3 and 4. Although Cluster 3 had a similar age as Cluster 2, it tended to have poorer outcomes. Both Clusters 3 and 4 tended to exhibit higher rates of oxygen supplementation, intensive care unit admission, and mechanical ventilation, but the mortality rate tended to be lower in Cluster 3. CONCLUSIONS: We have successfully performed the first phenotyping of COVID-19 patients in Japan, which is clinically useful in predicting important outcomes, despite the simplicity of the cluster analysis method that does not use complex variables.

Characteristics of hospitalized patients with COVID-19 during the first to fifth waves of infection: a report from the Japan COVID-19 Task Force.

BACKGROUND: We aimed to elucidate differences in the characteristics of patients with coronavirus disease 2019 (COVID-19) requiring hospitalization in Japan, by COVID-19 waves, from conventional strains to the Delta variant. METHODS: We used secondary data from a database and performed a retrospective cohort study that included 3261 patients aged ≥ 18 years enrolled from 78 hospitals that participated in the Japan COVID-19 Task Force between February 2020 and September 2021. RESULTS: Patients hospitalized during the second (mean age, 53.2 years [standard deviation {SD}, ± 18.9]) and fifth (mean age, 50.7 years [SD ± 13.9]) COVID-19 waves had a lower mean age than those hospitalized during the other COVID-19 waves. Patients hospitalized during the first COVID-19 wave had a longer hospital stay (mean, 30.3 days [SD ± 21.5], p < 0.0001), and post-hospitalization complications, such as bacterial infections (21.3%, p < 0.0001), were also noticeable. In addition, there was an increase in the use of drugs such as remdesivir/baricitinib/tocilizumab/steroids during the latter COVID-19 waves. In the fifth COVID-19 wave, patients exhibited a greater number of presenting symptoms, and a higher percentage of patients required oxygen therapy at the time of admission. However, the percentage of patients requiring invasive mechanical ventilation was the highest in the first COVID-19 wave and the mortality rate was the highest in the third COVID-19 wave. CONCLUSIONS: We identified differences in clinical characteristics of hospitalized patients with COVID-19 in each COVID-19 wave up to the fifth COVID-19 wave in Japan. The fifth COVID-19 wave was associated with greater disease severity on admission, the third COVID-19 wave had the highest mortality rate, and the first COVID-19 wave had the highest percentage of patients requiring mechanical ventilation.

Development and evaluation of a rapid and cost-efficient NGS-based MHC class I genotyping method for macaques by using a prevalent short-read sequencer.

Rhesus macaque is one of the most widely used primate model animals for immunological research of infectious diseases including human immunodeficiency virus (HIV) infection. It is well known that major histocompatibility complex (MHC) class I genotypes affect the susceptibility and disease progression to simian immunodeficiency virus (SIV) in rhesus macaques, which is resembling to HIV in humans. It is required to convincingly determine the MHC genotypes in the immunological investigations, that is why several next-generation sequencing (NGS)-based methods have been established. In general, NGS-based genotyping methods using short amplicons are not often applied to MHC because of increasing number of alleles and inevitable ambiguity in allele detection, although there is an advantage of short read sequencing systems that are commonly used today. In this study, we developed a new high-throughput NGS-based genotyping method for MHC class I alleles in rhesus macaques and cynomolgus macaques. By using our method, 95% and 100% of alleles identified by PCR cloning-based method were detected in rhesus macaques and cynomolgus macaques, respectively, which were highly correlated with their expression levels. It was noted that the simulation of new-allele detection step using artificial alleles differing by a few nucleotide sequences from a known allele could be identified with high accuracy and that we could detect a real novel allele from a rhesus macaque sample. These findings supported that our method could be adapted for primate animal models such as macaques to reduce the cost and labor of previous NGS-based MHC genotyping.

SARS-CoV-2 R.1 lineage variants that prevailed in Tokyo in March 2021.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, such as B.1.1.7 and B.1.351, has become a crucial issue worldwide. Therefore, we began testing all patients with COVID-19 for the N501Y and E484K mutations by using polymerase chain reaction (PCR)-based methods. Nasopharyngeal swab samples from 108 patients who visited our hospital between February and April 2021 were analyzed. The samples were analyzed using reverse transcription-PCR with melting curve analysis to detect the N501Y and E484K mutations. A part of the samples was also subjected to whole-genome sequencing (WGS). Clinical parameters such as mortality and admission to the intensive care unit were analyzed to examine the association between increased disease severity and the E484K mutation. The ratio of cases showing the 501N + 484K mutation rapidly increased from 8% in February to 46% in March. WGS revealed that the viruses with 501N + 484K mutation are R.1 lineage variants. Evidence of increased disease severity related to the R.1 variants was not found. We found that the R.1 lineage variants rapidly prevailed in Tokyo in March 2021, which suggests the increased transmissibility of R.1 variants, while they showed no increased severity.

A Validation Study of the Mayo Clinic Phenotype-Based Genetic Test Prediction Score for Japanese Patients With Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a primary myocardial disorder with an autosomal-dominant disorder mainly caused by mutations in sarcomere genes. Recently, a phenotype-based genetic test prediction score for patients with HCM was introduced by Mayo Clinic. The genotype score was derived on the basis of the predictive effect of 6 clinical markers, and the total score was shown to be correlated with the yield of genetic testing. However, it has not been determined whether this prediction model is useful in Japanese HCM patients.Methods and Results:The utility of the Mayo Clinic HCM genotype predictor score in 209 Japanese unrelated patients with a clinical diagnosis of HCM who had undergone genetic testing for 6 sarcomere genes was assessed. Overall, 55 patients (26%) had pathogenic or likely pathogenic variants (60% being genotype-positive in familial cases). We divided the patients into 6 groups (groups with scores of from -1 to 5) according to the prediction score. The yields of genetic testing in the groups with scores of -1, 0, 1, 2, 3, 4, and 5 were 8%, 16%, 24%, 48%, 50%, 100%, and 89%, respectively, with an incremental increase in yield between each of the score subgroups (P<0.001). CONCLUSIONS: The Mayo Clinic HCM genotype predictor score is useful for predicting a positive genetic test result in Japanese HCM Patients.

Genetic determinants and an epistasis of LILRA3 and HLA-B*52 in Takayasu arteritis.

Takayasu arteritis (TAK) is a systemic vasculitis with severe complications that affects the aorta and its large branches. HLA-B*52 is an established susceptibility locus to TAK. To date, there are still only a limited number of reports concerning non-HLA susceptibility loci to TAK. We conducted a genome-wide association study (GWAS) and a follow-up study in a total of 633 TAK cases and 5,928 controls. A total of 510,879 SNPs were genotyped, and 5,875,450 SNPs were imputed together with HLA-B*52. Functional annotation of significant loci, enhancer enrichment, and pathway analyses were conducted. We identified four unreported significant loci, namely rs2322599, rs103294, rs17133698, and rs1713450, in PTK2B, LILRA3/LILRB2, DUSP22, and KLHL33, respectively. Two additional significant loci unreported in non-European GWAS were identified, namely HSPA6/FCGR3A and chr21q.22. We found that a single variant associated with the expression of MICB, a ligand for natural killer (NK) cell receptor, could explain the entire association with the HLA-B region. Rs2322599 is strongly associated with the expression of PTK2B Rs103294 risk allele in LILRA3/LILRB2 is known to be a tagging SNP for the deletion of LILRA3, a soluble receptor of HLA class I molecules. We found a significant epistasis effect between HLA-B*52 and rs103294 (P = 1.2 × 10-3). Enhancer enrichment analysis and pathway analysis suggested the involvement of NK cells (P = 8.8 × 10-5, enhancer enrichment). In conclusion, four unreported TAK susceptibility loci and an epistasis effect between LILRA3 and HLA-B*52 were identified. HLA and non-HLA regions suggested a critical role for NK cells in TAK.

A Novel Titin Truncation Variant Linked to Familial Dilated Cardiomyopathy Found in a Japanese Family and Its Functional Analysis in Genome-Edited Model Cells.

Dilated cardiomyopathy (DCM) is a common cause of heart failure. TTN, which encodes titin protein, is a representative causative gene of DCM, and is presented mainly as a truncation variant. However, TTN truncation variants are also found in healthy individuals, and it is therefore important to evaluate the pathogenicity of each variant. In this study, we analyzed 67 cardiomyopathy-associated genes in a male Japanese patient who was hospitalized for recurrent severe heart failure and identified a novel truncation variant, TTN Ser17456Arg fs*14. This TTN truncation variant was located in the A-band region. Moreover, the patient's mother with heart failure harbored the same variant, whereas the father and brother without heart failure did not harbor the variant. To examine the functional changes associated with the truncation variant, H9c2 cells were subjected to genome editing to generate cells with a homologous truncation variant. The cells were differentiated using all-trans-retinoic acid, and the mRNA expression of skeletal actin and cardiac actin were found to be increased and decreased, respectively, consistent with known changes in patients with DCM or heart failure. In contrast, another cell with the titin truncation variant used as a control showed no changes in heart failure-related genes. In summary, we found a novel TTN truncation variant in familial DCM patients and confirmed its functional changes using a relatively simple cell model. The novel truncation variant was identified as a pathogenic and disease-causing mutation.

Histopathological changes of myocytes in restrictive cardiomyopathy.

Restrictive cardiomyopathy (RCM) is a rare primary myocardial disease, and its pathological features are yet to be determined. Restrictive cardiomyopathy with MHY7 mutation was diagnosed in a 65-year-old Japanese woman. Electron microscopy of a myocardial biopsy revealed electron-dense materials resulting from focal myocyte degeneration and necrosis as well as tubular structures and pseudo-inclusion bodies in some nuclei. These features may be associated with the pathogenesis of RCM.

Determination of a T cell receptor of potent CD8+ T cells against simian immunodeficiency virus infection in Burmese rhesus macaques.

Cumulative studies on human immunodeficiency virus (HIV)-infected individuals have shown association of major histocompatibility complex class I (MHC-I) polymorphisms with lower viral load and delayed AIDS progression, suggesting that HIV replication can be controlled by potent CD8+ T-cell responses. We have previously established an AIDS model of simian immunodeficiency virus (SIV) infection in Burmese rhesus macaques and found a potent CD8+ T cell targeting the Mamu-A1*065:01-restricted Gag241-249 epitope, which is located in a region corresponding to the HIV Gag240-249 TW10 epitope restricted by a protective MHC-I allele, HLA-B*57. In the present study, we determined a T cell receptor (TCR) of this Gag241-249 epitope-specific CD8+ T cell. cDNA clones encoding TCR-α and TCR-ß chains were obtained from a Gag241-249-specific CD8+ T-cell clone. Coexpression of these TCR-α and TCR-ß cDNAs resulted in reconstitution of a functional TCR specifically detected by Gag241-249 epitope-Mamu-A1*065:01 tetramer. Two of three previously-reported CD8+ T-cell escape mutations reduced binding affinity of Gag241-249 peptide to Mamu-A1*065:01 but the remaining one not. This is consistent with the data obtained by molecular modeling of the epitope-MHC-I complex and TCR. These results would contribute to understanding how viral CD8+ T-cell escape mutations are selected under structural constraint of viral proteins.

Lifelong Clinical Impact of the Presence of Sarcomere Gene Mutation in Japanese Patients With Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is mainly caused by mutations in sarcomere genes. Regarding the clinical implications of genetic information, little is known about the lifelong clinical effect of sarcomere mutations in Japanese HCM patients.Methods and Results:We studied 211 consecutive Japanese patients with HCM who had agreed to genetic testing between 2003 and 2013. Genetic analyses were performed by direct DNA sequencing in the 6 common sarcomere genes (MYH7,MYBPC3,TNNT2,TNNI3,TPM1,ACTC). Through variant filtering, 21 mutations were identified in 67 patients. After excluding 8 patients whose variants were determined as having uncertain significance, finally 203 patients (130 men, age at study entry: 61.8±14.1 years) were investigated for clinical presentation and course. At the time of study entry, patients with mutations were younger, had more frequent non-sustained ventricular tachycardia, had greater interventricular wall thickness, were more frequently in the dilated phase and less frequently had apical HCM. Through their lifetimes, a total of 98 HCM-related morbid events occurred in 72 patients. Survival analysis revealed that patients with sarcomere gene mutations experienced those morbid events significantly more frequently, and this tendency was more prominent for lethal arrhythmic events. CONCLUSIONS: In our HCM cohort, patients with sarcomere gene mutations had poorer lifelong outcome. Genetic information is considered important for better management of HCM.