Long-read sequencing identifies an SVA_D retrotransposon insertion deep within the intron of ATP7A as a novel cause of occipital horn syndrome.

BACKGROUND: SINE-VNTR-Alu (SVA) retrotransposons move from one genomic location to another in a 'copy-and-paste' manner. They continue to move actively and cause monogenic diseases through various mechanisms. Currently, disease-causing SVA retrotransposons are classified into human-specific young SVA_E or SVA_F subfamilies. In this study, we identified an evolutionarily old SVA_D retrotransposon as a novel cause of occipital horn syndrome (OHS). OHS is an X-linked, copper metabolism disorder caused by dysfunction of the copper transporter, ATP7A. METHODS: We investigated a 16-year-old boy with OHS whose pathogenic variant could not be detected via routine molecular genetic analyses. RESULTS: A 2.8 kb insertion was detected deep within the intron of the patient's ATP7A gene. This insertion caused aberrant mRNA splicing activated by a new donor splice site located within it. Long-read circular consensus sequencing enabled us to accurately read the entire insertion sequence, which contained highly repetitive and GC-rich segments. Consequently, the insertion was identified as an SVA_D retrotransposon. Antisense oligonucleotides (AOs) targeting the new splice site restored the expression of normal transcripts and functional ATP7A proteins. AO treatment alleviated excessive accumulation of copper in patient fibroblasts in a dose-dependent manner. Pedigree analysis revealed that the retrotransposon had moved into the OHS-causing position two generations ago. CONCLUSION: This is the first report of a human monogenic disease caused by the SVA_D retrotransposon. The fact that the evolutionarily old SVA_D is still actively transposed, leading to increased copy numbers may make a notable impact on rare genetic disease research.

Blue Wavelength of Scanning Laser Ophthalmoscope Potentially Detects Arteriosclerotic Lesions in Diabetic Retinopathy.

(1) Background: The fundus examination is one of the best and popular methods in the assessment of vascular status in the human body. Direct viewing of retinal vessels by ophthalmoscopy has been utilized in judging hypertensive change or arteriosclerosis. Recently, fundus imaging with the non-mydriatic scanning laser ophthalmoscope (SLO) has been widely used in ophthalmological clinics since it has multimodal functions for optical coherence tomography or angiography with contrast agent dye. The purpose of this study was to examine the utility in detecting arteriosclerosis of retinal vessels in SLO images; (2) Methods: Both color and blue standard field SLO images of eyes with diabetic retinopathy (DR) were examined retrospectively. Retinal arteriosclerosis in color SLO images was graded according to the Scheie classification. Additionally, characteristics of retinal arterioles in blue SLO images were identified and examined for their relevance to arteriosclerosis grades, stages of DR or general complications; (3) Results: Relative to color fundus images, blue SLO images showed distinct hyper-reflective retinal arterioles against a monotone background. Irregularities of retinal arterioles identified in blue SLO images were frequently observed in the eyes of patients with severe arteriosclerosis (Grade 3: 79.0% and Grade 4: 81.8%). Furthermore, the findings on arterioles were more frequently associated with the eyes of DR patients with renal dysfunction (p < 0.05); (4) Conclusions: While color SLO images are equally as useful in assessing retinal arteriosclerosis as photography or ophthalmoscopy, the corresponding blue SLO images show arteriosclerotic lesions with high contrast in a monotone background. Retinal arteriosclerosis in eyes of advanced grades or advanced DR frequently show irregularities of retinal arterioles in the blue images. The findings of low, uneven, or discontinuous attenuation were easier to find in blue than in color SLO images. Consequently, blue SLO images can show pathological micro-sclerosis in retinal arterioles and are potentially one of the safe and practical methods for the vascular assessment of diabetic patients.

Genetic Analysis of SCN11A, SCN10A, and SCN9A in Familial Episodic Pain Syndrome (FEPS) in Japan and Proposal of Clinical Diagnostic Criteria.

Familial episodic pain syndrome (FEPS) is an early childhood onset disorder of severe episodic limb pain caused mainly by pathogenic variants of SCN11A, SCN10A, and SCN9A, which encode three voltage-gated sodium channels (VGSCs) expressed as key determinants of nociceptor excitability in primary sensory neurons. There may still be many undiagnosed patients with FEPS. A better understanding of the associated pathogenesis, epidemiology, and clinical characteristics is needed to provide appropriate diagnosis and care. For this study, nationwide recruitment of Japanese patients was conducted using provisional clinical diagnostic criteria, followed by genetic testing for SCN11A, SCN10A, and SCN9A. In the cohort of 212 recruited patients, genetic testing revealed that 64 patients (30.2%) harbored pathogenic or likely pathogenic variants of these genes, consisting of 42 (19.8%), 14 (6.60%), and 8 (3.77%) patients with variants of SCN11A, SCN10A, and SCN9A, respectively. Meanwhile, the proportions of patients meeting the tentative clinical criteria were 89.1%, 52.0%, and 54.5% among patients with pathogenic or likely pathogenic variants of each of the three genes, suggesting the validity of these clinical criteria, especially for patients with SCN11A variants. These clinical diagnostic criteria of FEPS will accelerate the recruitment of patients with underlying pathogenic variants who are unexpectedly prevalent in Japan.

A 37-Year-Old Man With Intellectual Disability Discovered to Have Aspartylglucosaminuria: Implications for the Diagnosis of Genetic Causes.

Objectives: The causes of intellectual disability (ID) are varied, with as many as 1,400 causative genes. We attempted to identify the causative gene in a patient with long-standing undiagnosed ID. Methods: Although this was an isolated case with no family history, we searched for the causative gene using trio-based whole-exome sequencing (trio-WES), because severe ID is often caused by genetic variations, and inherited metabolic disorders (IMDs) are assumed to be the cause when regression and epilepsy occur. Results: We identified homozygous donor splice-site variants in the AGA gene (aspartylglucosaminidase; NM_000027.4) Chr4(GRCh38):g. 177436275C>A, c.698+1G>T. This gene is implicated in aspartylglucosaminuria (AGU; OMIM #208400) and originated from both of the patient's parents. We confirmed the pathogenicity of the variant by detecting the splicing defect in cDNA from the patient's blood and accumulation of aberrant metabolites in the patient's urine. Discussion: We discuss how to more readily achieve an accurate diagnosis for patients with undiagnosed intellectual disabilities. Medical practitioners' awareness of the characteristics of the disease leading to clinical suspicion in patients with matching presentations, and the performance of newborn screening when possible, is important for the diagnosis of ID. In addition, the characteristic symptoms and course of the disease give rise to suspicion of IMDs. Given our results, we consider trio-WES to be a powerful method for identifying the causative genes in cases of ID with genetic causes.

Prophylactic vaccination inducing anti-Env antibodies can result in protection against HTLV-1 challenge in macaques.

Human T cell leukemia/T-lymphotropic virus type 1 (HTLV-1) infection occurs by cell-to-cell transmission and can induce fatal adult T cell leukemia. Vaccine development is critical for the control of HTLV-1 transmission. However, determining whether vaccine-induced anti-Env antibodies can prevent cell-to-cell HTLV-1 transmission is challenging. Here, we examined the protective efficacy of a vaccine inducing anti-Env antibodies against HTLV-1 challenge in cynomolgus macaques. Eight of 10 vaccinated macaques produced anti-HTLV-1 neutralizing antibodies (NAbs) and were protected from an intravenous challenge with 108 HTLV-1-producing cells. In contrast, the 2 vaccinated macaques without NAb induction and 10 unvaccinated controls showed HTLV-1 infection with detectable proviral load after challenge. Five of the eight protected macaques were administered with an anti-CD8 monoclonal antibody, but proviruses remained undetectable and no increase in anti-HTLV-1 antibodies was observed even after CD8+ cell depletion in three of them. Analysis of Env-specific T cell responses did not suggest involvement of vaccine-induced Env-specific T cell responses in the protection. These results indicate that anti-Env antibody induction by vaccination can result in functionally sterile HTLV-1 protection, implying the rationale for strategies aimed at anti-Env antibody induction in prophylactic HTLV-1 vaccine development.

Juvenile xanthogranuloma manifesting with LCH-associated neurodegenerative disease-like radiological findings.

Here, we describe two patients with juvenile xanthogranuloma (JXG) manifesting with Langerhans cell histiocytosis (LCH)-associated neurodegenerative disease (ND)-like radiological findings. One patient showed typical radiological abnormalities at onset, which worsened with progressing central nervous system symptoms 7 years after LCH-oriented chemotherapy. Another showed spontaneous regression of clinical symptoms, with a transient radiological change 1 year after salvage chemotherapy for recurrence of JXG. These data regarding JXG-associated ND will facilitate future investigation of the disease, as well as development of therapeutic interventions.

Single-Cell RNA Sequencing Reveals an Immune Landscape of CD4+ T Cells in Coronary Culprit Plaques With Acute Coronary Syndrome in Humans-Brief Report.

BACKGROUND: Acute coronary syndrome (ACS) involves plaque-related thrombosis, causing primary ischemic cardiomyopathy or lethal arrhythmia. We previously demonstrated a unique immune landscape of myeloid cells in the culprit plaques causing ACS by using single-cell RNA sequencing. Here, we aimed to characterize T cells in a single-cell level, assess clonal expansion of T cells, and find a therapeutic target to prevent ACS. METHODS: We obtained the culprit lesion plaques from 4 patients with chronic coronary syndrome (chronic coronary syndrome plaques) and the culprit lesion plaques from 3 patients with ACS (ACS plaques) who were candidates for percutaneous coronary intervention with directional coronary atherectomy. Live CD45+ immune cells were sorted from each pooled plaque samples and applied to the 10× platform for single-cell RNA sequencing analysis. We also extracted RNA from other 3 ACS plaque samples and conducted unbiased TCR (T-cell receptor) repertoire analysis. RESULTS: CD4+ T cells were divided into 5 distinct clusters: effector, naive, cytotoxic, CCR7+ (C-C chemokine receptor type 7) central memory, and FOXP3 (forkhead box P3)+ regulatory CD4+ T cells. The proportion of central memory CD4+ T cells was higher in the ACS plaques. Correspondingly, dendritic cells also tended to express more HLAs (human leukocyte antigens) and costimulatory molecules in the ACS plaques. The velocity analysis suggested the differentiation flow from central memory CD4+ T cells into effector CD4+ T cells and that from naive CD4+ T cells into central memory CD4+ T cells in the ACS plaques, which were not observed in the chronic coronary syndrome plaques. The bulk repertoire analysis revealed clonal expansion of TCRs in each patient with ACS and suggested that several peptides in the ACS plaques work as antigens and induced clonal expansion of CD4+ T cells. CONCLUSIONS: For the first time, we revealed single cell-level characteristics of CD4+ T cells in patients with ACS. CD4+ T cells could be therapeutic targets of ACS. REGISTRATION: URL: https://upload.umin.ac.jp/cgi-open-bin/icdr_e/ctr_view.cgi?recptno=R000046521; Unique identifier: UMIN000040747.

Soluble immune checkpoint factors reflect exhaustion of antitumor immunity and response to PD-1 blockade.

BACKGROUNDPrecise stratification of patients with non-small cell lung cancer (NSCLC) is needed for appropriate application of PD-1/PD-L1 blockade therapy.METHODSWe measured soluble forms of the immune-checkpoint molecules PD-L1, PD-1, and CTLA-4 in plasma of patients with advanced NSCLC before PD-1/PD-L1 blockade. A prospective biomarker-finding trial (cohort A) included 50 previously treated patients who received nivolumab. A retrospective observational study was performed for patients treated with any PD-1/PD-L1 blockade therapy (cohorts B and C), cytotoxic chemotherapy (cohort D), or targeted therapy (cohort E). Plasma samples from all patients were assayed for soluble immune-checkpoint molecules with a highly sensitive chemiluminescence-based assay.RESULTSNonresponsiveness to PD-1/PD-L1 blockade therapy was associated with higher concentrations of these soluble immune factors among patients with immune-reactive (hot) tumors. Such an association was not apparent for patients treated with cytotoxic chemotherapy or targeted therapy. Integrative analysis of tumor size, PD-L1 expression in tumor tissue (tPD-L1), and gene expression in tumor tissue and peripheral CD8+ T cells revealed that high concentrations of the 3 soluble immune factors were associated with hyper or terminal exhaustion of antitumor immunity. The combination of soluble PD-L1 (sPD-L1) and sCTLA-4 efficiently discriminated responsiveness to PD-1/PD-L1 blockade among patients with immune-reactive tumors.CONCLUSIONCombinations of soluble immune factors might be able to identify patients unlikely to respond to PD-1/PD-L1 blockade as a result of terminal exhaustion of antitumor immunity. Our data suggest that such a combination better predicts, along with tPD-L1, for the response of patients with NSCLC.TRIAL REGISTRATIONUMIN000019674.FUNDINGThis study was funded by Ono Pharmaceutical Co. Ltd. and Sysmex Corporation.

Temporal Protrusion of the Optic Disc Implicated in Central Visual Field Abnormalities in High Myopia: Two Case Reports.

Introduction: Severe central visual field defects are frequently observed in highly myopic eyes. This report details 2 cases of central visual field defects in individuals with high myopia, characterized by an unusual temporal protrusion of the optic disc, a feature not previously documented. Case Presentation: Two patients, a 54-year-old man and a 65-year-old woman, were diagnosed with high myopia in their left eyes, displaying an outward protrusion of the optic disc toward the macula. Swept-source optical coherence tomography revealed a focal lamina cribrosa defect at the temporal edge of the protruding optic disc, corresponding to the papillomacular bundle area of retinal nerve fibers, which exhibited thinning around the focal lamina cribrosa defect. Visual field examination indicated a central visual field defect in the affected eyes, which pattern corresponded to the papillomacular bundle responsible area. Conclusion: The emergence of a temporal protrusion in the optic disc may lead to a focal lamina cribrosa defect, resulting in a central visual field defect in highly myopic eyes. This distinctive optic disc feature may constitute a critical risk factor for a central visual field defect. Hence, optic disc protrusion in high myopia warrants attention, necessitating careful ophthalmic examinations for central visual field defects.

Combined Use of Alkaline Agents With Low-Flow Extracorporeal Carbon Dioxide Removal in Carbon Dioxide Inhalation Models Preserving Inspiratory Efforts.

Background: Low-flow extracorporeal CO 2 removal (ECCO 2 R), managed using a renal replacement platform, is useful in achieving lung-protective ventilation with low tidal volume. However, its capacity for CO 2 elimination is limited. Whether this system is valuable in reducing strong inspiratory efforts in respiratory failure is unclear. The combined use of alkaline agents with low-flow ECCO 2 R might be useful in hypercapnic subjects preserving inspiratory efforts. Methods: This study examined the effects of low-flow ECCO 2 R on respiratory status and investigated the effects of NaHCO 3 , trometamol, and saline on respiratory status during low-flow ECCO 2 R in CO 2 inhalation models. Results: Although low-flow ECCO 2 R did not significantly change the respiratory rate (92.2% ± 24.3% [mean ± standard deviation] of that before ECCO 2 R), it reduced minute ventilation (MV) (78.9% ± 13.5% of that before ECCO 2 R). The addition of NaHCO 3 improved acidemia but did not change MV compared with that of the saline group (0.451 ± 0.026 L/min/kg body weight [BW] vs. 0.556 ± 0.138 L/min/kg BW, respectively). The addition of trometamol improved acidemia and reduced MV compared with that of the saline group (0.381 ± 0.050 L/min/kg BW vs. 0.556 ± 0.138 L/min/kg BW, respectively). The total amounts of CO 2 removed during ECCO 2 R in the NaHCO 3 group were lower than those in the saline and trometamol groups. Conclusion: The low-flow ECCO 2 R reduced MV in subjects preserving spontaneous breathing efforts with CO 2 overload. The addition of NaHCO 3 improved acidemia but did not change MV, whereas the addition of trometamol improved acidemia and reduced MV.

Precise analysis of single small extracellular vesicles using flow cytometry.

Methods that enable specific and sensitive quantification of small extracellular vesicles (sEVs) using flow cytometry are still under development. Aggregation or adsorption of antibodies causes sub-nano sized particles or non-specific binding and largely affects the results of flow cytometric analysis of single sEVs. Comparison of control IgG and target-specific IgG is inappropriate because they have different characters. Here, we evaluate four preparation methods for flow cytometry, including ultracentrifugation, density gradient centrifugation, size exclusion chromatography (SEC), and the TIM4-affinity method by using tetraspanin-deficient sEVs. The ultracentrifugation or density gradient centrifugation preparation method has large false-positive rates for tetraspanin staining. Conversely, preparation methods using SEC or the TIM4-affinity method show specific detection of single sEVs, which elucidate the roles of sEV biogenesis regulators in the generation of sEV subpopulations. The methods are also useful for the detection of rare disease-related markers, such as PD-L1. Flow cytometric analysis using SEC or the TIM4-affinity method could accelerate research into sEV biogenesis and the development of sEV-based diagnostics and therapies.

Extracellular vesicles promote silica nanoparticle aggregation that inhibits silica-induced cytotoxicity.

Amorphous silica has been approved as a food and pharmaceutical additive. However, its potential to enhance the carcinogenicity of epithelial cells is incontrovertible. With their expanded surface area per unit mass and distinctive cellular incorporation, nano-sized silica particles (nSPs) exhibit heightened cytotoxicity compared to micrometer-sized counterparts. The precise effect of nSPs on the generation of small extracellular vesicles (sEVs) within endosomes after cellular uptake remains unclear. In the present study, we explored the secretion of sEVs from cells and their functional implications following exposure to nSPs. Our findings demonstrate that nSP50 exposure not only induced epithelial-mesenchymal transition (EMT) but also promoted the maturation of multivesicular endosomes (MVEs) along with the secretion of sEVs in A549 cells. Inhibition of sEV secretion using GW4869 and apoptosis activator 2 exacerbated nSP50-induced EMT, indicating that sEV secretion may suppress EMT. Analysis of the function of sEV in a cell-free system revealed that co-incubation of sEVs with nSP50 led to the formation of micrometer-sized aggregates, which exhibited limited uptake efficiency within A549 cells. These results strongly suggest that the secretion of sEVs plays a protective role against the cytotoxicity attributed to nSP50 exposure.

Specific consistency score for rational selection of epilepsy resection surgery candidates.

OBJECTIVE: Degree of indication for epilepsy surgery is determined by taking multiple factors into account. This study aimed to investigate the usefulness of the Specific Consistency Score (SCS), a proposed score for focal epilepsy to rate the indication for epilepsy focal resection. METHODS: This retrospective cohort study included patients considered for resective epilepsy surgery in Kyoto University Hospital from 2011 to 2022. Plausible epileptic focus was tentatively defined. Cardinal findings were scored based on specificity and consistency with the estimated laterality and lobe. The total points represented SCS. The association between SCS and the following clinical parameters was assessed by univariate and multivariate analysis: (1) probability of undergoing resective epilepsy surgery, (2) good postoperative seizure outcome (Engel I and II or Engel I only), and (3) lobar concordance between the noninvasively estimated focus and intracranial electroencephalographic (EEG) recordings. RESULTS: A total of 131 patients were evaluated. Univariate analysis revealed higher SCS in the (1) epilepsy surgery group (8.4 [95% confidence interval (CI) = 7.8-8.9] vs. 4.9 [95% CI = 4.3-5.5] points; p < .001), (2) good postoperative seizure outcome group (Engel I and II; 8.7 [95% CI = 8.2-9.3] vs. 6.4 [95% CI = 4.5-8.3] points; p = .008), and (3) patients whose focus defined by intracranial EEG matched the noninvasively estimated focus (8.3 [95% CI = 7.3-9.2] vs. 5.4 [95% CI = 3.5-7.3] points; p = .004). Multivariate analysis revealed areas under the curve of .843, .825, and .881 for Parameters 1, 2, and 3, respectively. SIGNIFICANCE: SCS provides a reliable index of good indication for resective epilepsy surgery and can be easily available in many institutions not necessarily specializing in epilepsy.

bmp-2 Gene-Transferred Skeletal Muscles with Needle-Type Electrodes as Efficient and Reliable Biomaterials for Bone Regeneration.

BACKGROUND: Bone morphogenetic protein-2 (bmp-2) has a high potential to induce bone tissue formation in skeletal muscles. We developed a bone induction system in skeletal muscles using the bmp-2 gene through in vivo electroporation. Natural bone tissues with skeletal muscles can be considered potential candidates for biomaterials. However, our previous system using plate-type electrodes did not achieve a 100% success rate in inducing bone tissues in skeletal muscles. In this study, we aimed to enhance the efficiency of bone tissue formation in skeletal muscles by using a non-viral bmp-2 gene expression plasmid vector (pCAGGS-bmp-2) and needle-type electrodes. METHODS: We injected the bmp-2 gene with pCAGGS-bmp-2 into the skeletal muscles of rats' legs and immediately placed needle-type electrodes there. Skeletal tissues were then observed on the 21st day after gene transfer using soft X-ray and histological analyses. RESULTS: The use of needle-type electrodes resulted in a 100% success rate in inducing bone tissues in skeletal muscles. In contrast, the plate-type electrodes only exhibited a 33% success rate. Thus, needle-type electrodes can be more efficient and reliable for transferring the bmp-2 gene to skeletal muscles, making them potential biomaterials for repairing bone defects.

Electrical Impedance Tomography-based Ventilation Patterns in Patients after Major Surgery.

Rationale: General anesthesia and mechanical ventilation have negative impacts on the respiratory system, causing heterogeneous distribution of lung aeration, but little is known about the ventilation patterns of postoperative patients and their association with clinical outcomes. Objectives: To clarify the phenotypes of ventilation patterns along a gravitational direction after surgery by using electrical impedance tomography (EIT) and to evaluate their association with postoperative pulmonary complications (PPCs) and other relevant clinical outcomes. Methods: Adult postoperative patients at high risk for PPCs, receiving mechanical ventilation on ICU admission (N = 128), were prospectively enrolled between November 18, 2021 and July 18, 2022. PPCs were prospectively scored until hospital discharge, and their association with phenotypes of ventilation patterns was studied. The secondary outcomes were the times to wean from mechanical ventilation and oxygen use and the length of ICU stay. Measurements and Main Results: Three phenotypes of ventilation patterns were revealed by EIT: phenotype 1 (32% [n = 41], a predominance of ventral ventilation), phenotype 2 (41% [n = 52], homogeneous ventilation), and phenotype 3 (27% [n = 35], a predominance of dorsal ventilation). The median PPC score was higher in phenotype 1 and phenotype 3 than in phenotype 2. The median time to wean from mechanical ventilation was longer in phenotype 1 versus phenotype 2. The median duration of ICU stay was longer in phenotype 1 versus phenotype 2. The median time to wean from oxygen use was longer in phenotype 1 and phenotype 3 than in phenotype 2. Conclusions: Inhomogeneous ventilation patterns revealed by EIT on ICU admission were associated with PPCs, delayed weaning from mechanical ventilation and oxygen use, and a longer ICU stay.