Dissecting the genetic and causal relationship between sleep-related traits and common brain disorders.

BACKGROUND: There is a profound connection between abnormal sleep patterns and brain disorders, suggesting a shared influential association. However, the shared genetic basis and potential causal relationships between sleep-related traits and brain disorders are yet to be fully elucidated. METHODS: Utilizing linkage disequilibrium score regression (LDSC) and bidirectional two-sample univariable Mendelian Randomization (UVMR) analyses with large-scale GWAS datasets, we investigated the genetic correlations and causal associations across six sleep traits and 24 prevalent brain disorders. Additionally, a multivariable Mendelian Randomization (MVMR) analysis evaluated the cumulative effects of various sleep traits on each brain disorder, complemented by genetic loci characterization to pinpoint pertinent genes and pathways. RESULTS: LDSC analysis identified significant genetic correlations in 66 out of 144 (45.8 %) pairs between sleep-related traits and brain disorders, with the most pronounced correlations observed in psychiatric disorders (66 %, 48/72). UVMR analysis identified 29 causal relationships (FDR<0.05) between sleep traits and brain disorders, with 19 associations newly discovered according to our knowledge. Notably, major depression, attention-deficit/hyperactivity disorder, bipolar disorder, cannabis use disorder, and anorexia nervosa showed bidirectional causal relations with sleep traits, especially insomnia's marked influence on major depression (IVW beta 0.468, FDR = 5.24E-09). MVMR analysis revealed a nuanced interplay among various sleep traits and their impact on brain disorders. Genetic loci characterization underscored potential genes, such as HOXB2, while further enrichment analyses illuminated the importance of synaptic processes in these relationships. CONCLUSIONS: This study provides compelling evidence for the causal relationships and shared genetic backgrounds between common sleep-related traits and brain disorders.

Lapatinib combined with doxorubicin causes dose-dependent cardiotoxicity partially through activating the p38MAPK signaling pathway in zebrafish embryos.

Because of its enhanced antitumor efficacy, lapatinib (LAP) is commonly used clinically in combination with the anthracycline drug doxorubicin (DOX) to treat metastatic breast cancer. While it is well recognized that this combination chemotherapy can lead to an increased risk of cardiotoxicity in adult women, its potential cardiotoxicity in the fetus during pregnancy remains understudied. Here, we aimed to examine the combination of LAP chemotherapy and DOX-induced cardiotoxicity in the fetus using a zebrafish embryonic system and investigate the underlying pathologic mechanisms. First, we examined the dose-dependent cardiotoxicity of combined LAP and DOX exposure in zebrafish embryos, which mostly manifested as pericardial edema, bradycardia, cardiac function decline and reduced survival. Second, we revealed that a significant increase in oxidative stress concurrent with activated MAPK signaling, as indicated by increased protein expression of phosphorylated p38 and Jnk, was a notable pathophysiological event after combined LAP and DOX exposure. Third, we showed that inhibiting MAPK signaling by pharmacological treatment with the p38MAPK inhibitor SB203580 or genetic ablation of the map2k6 gene could significantly alleviate combined LAP and DOX exposure-induced cardiotoxicity. Thus, we provided both pharmacologic and genetic evidence to suggest that inhibiting MAPK signaling could exert cardioprotective effects. These findings have implications for understanding the potential cardiotoxicity induced by LAP and DOX combinational chemotherapy in the fetus during pregnancy, which could be leveraged for the development of new therapeutic strategies.

FAdV-4-induced ferroptosis affects fat metabolism in LMH cells.

Ferroptosis is a form of controlled cell death that was first described relatively recently and that is dependent on the formation and accumulation of lipid free radicals through an iron-mediated mechanism. A growing body of evidence supports the close relationship between pathogenic infections and ferroptotic cell death, particularly for viral infections. Ferroptosis is also closely tied to the pathogenic development of hepatic steatosis and other forms of liver disease. Fowl adenovirus serotype 4 (FAdV-4) is a hepatotropic aviadenovirus causing hydropericardium syndrome (HPS) that is capable of impacting fat metabolism. However, it remains uncertain as to what role, if any, ferroptotic death plays in the context of FAdV-4 infection. Here, FAdV-4 was found to promote ferroptosis via the p53-SLC7A11-GPX4 axis, while ferrostain-1 was capable of inhibiting this FAdV-4-mediated ferroptotic death through marked reductions in lipid peroxidation. The incidence of FAdV-4-induced fatty liver was also found to be associated with the activation of ferroptotic activity. Together, these results offer novel insights regarding potential approaches to treating HPS.

Genome-wide investigation and expression profiling of LOR gene family in rapeseed under salinity and ABA stress.

The Brassica napus (B. napus) LOR (Lurp-One-Related) gene family is a little-known gene family characterized by a conserved LOR domain in the proteins. Limited research in Arabidopsis showed that LOR family members played important roles in Hyaloperonospora parasitica (Hpa) defense. Nevertheless, there is a paucity of research investigating the role of the LOR gene family towards their responses to abiotic stresses and hormone treatments. This study encompassed a comprehensive survey of 56 LOR genes in B. napus, which is a prominent oilseed crop that holds substantial economic significance in China, Europe, and North America. Additionally, the study evaluated the expression profiles of these genes in response to salinity and ABA stress. Phylogenetic analysis showed that 56 BnLORs could be divided into 3 subgroups (8 clades) with uneven distribution on 19 chromosomes. 37 out of 56 BnLOR members have experienced segmental duplication and 5 of them have undergone tandem repeats events with strong evidence of purifying selection. Cis-regulatory elements (CREs) analysis indicated that BnLORs involved in process such as light response, hormone response, low temperature response, heat stress response, and dehydration response. The expression pattern of BnLOR family members revealed tissue specificity. RNA-Seq and qRT-PCR were used to validate BnLOR gene expression under temperature, salinity and ABA stress, revealing that most BnLORs showed inducibility. This study enhanced our comprehension of the B. napus LOR gene family and could provide valuable information for identifying and selecting genes for stress resistant breeding.

Using Zebrafish Animal Model to Study the Genetic Underpinning and Mechanism of Arrhythmogenic Cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is largely an autosomal dominant genetic disorder manifesting fibrofatty infiltration and ventricular arrhythmia with predominantly right ventricular involvement. ACM is one of the major conditions associated with an increased risk of sudden cardiac death, most notably in young individuals and athletes. ACM has strong genetic determinants, and genetic variants in more than 25 genes have been identified to be associated with ACM, accounting for approximately 60% of ACM cases. Genetic studies of ACM in vertebrate animal models such as zebrafish (Danio rerio), which are highly amenable to large-scale genetic and drug screenings, offer unique opportunities to identify and functionally assess new genetic variants associated with ACM and to dissect the underlying molecular and cellular mechanisms at the whole-organism level. Here, we summarize key genes implicated in ACM. We discuss the use of zebrafish models, categorized according to gene manipulation approaches, such as gene knockdown, gene knock-out, transgenic overexpression, and CRISPR/Cas9-mediated knock-in, to study the genetic underpinning and mechanism of ACM. Information gained from genetic and pharmacogenomic studies in such animal models can not only increase our understanding of the pathophysiology of disease progression, but also guide disease diagnosis, prognosis, and the development of innovative therapeutic strategies.

Application of an F0-based genetic assay in adult zebrafish to identify modifier genes of an inherited cardiomyopathy.

Modifier genes contribute significantly to our understanding of pathophysiology in human diseases; however, effective approaches to identify modifier genes are still lacking. Here, we aim to develop a rapid F0-based genetic assay in adult zebrafish using the bag3 gene knockout (bag3e2/e2) cardiomyopathy model as a paradigm. First, by utilizing a classic genetic breeding approach, we identified dnajb6b as a deleterious modifier gene for bag3 cardiomyopathy. Next, we established an F0-based genetic assay in adult zebrafish through injection of predicted microhomology-mediated end joining (MMEJ)-inducing single guide RNA/Cas9 protein complex. We showed that effective gene knockdown is maintained in F0 adult fish, enabling recapitulation of both salutary modifying effects of the mtor haploinsufficiency and deleterious modifying effects of the dnajb6b gene on bag3 cardiomyopathy. We finally deployed the F0-based genetic assay to screen differentially expressed genes in the bag3 cardiomyopathy model. As a result, myh9b was identified as a novel modifier gene for bag3 cardiomyopathy. Together, these data prove the feasibility of an F0 adult zebrafish-based genetic assay that can be effectively used to discover modifier genes for inherited cardiomyopathy.

Cinnamaldehyde causes developmental neurotoxicity in zebrafish via the oxidative stress pathway that is rescued by astaxanthin.

Toxicology studies provide a reliable dose range for the use of compounds. Zebrafish show unique advantages in toxicology research. Cinnamaldehyde (Cin) is one of the main active compounds isolated from Cinnamon trees and other species of the genus Cinnamomum. In this study, we investigated the developmental neurotoxicity of cinnamaldehyde in zebrafish and preliminarily explored its underlying mechanism. Cinnamaldehyde causes developmental neurotoxicity in zebrafish, as evidenced by the damage to ventricular structures, eye malformations, shortened body length, trunk curvature, decreased neuronal fluorescence, and pericardial oedema. Moreover, it can induce abnormal behaviour and gene expression in zebrafish. After treatment with the oxidative stress inhibitor astaxanthin, the behaviour and abnormal gene expression were reversed. All of these data demonstrated that the developmental neurotoxicity of cinnamaldehyde might be attributed to oxidative stress. In addition, this study also confirmed that zebrafish is a reliable model for toxicity studies.

Genetic therapy in a mitochondrial disease model suggests a critical role for liver dysfunction in mortality.

The clinical and largely unpredictable heterogeneity of phenotypes in patients with mitochondrial disorders demonstrates the ongoing challenges in the understanding of this semi-autonomous organelle in biology and disease. Previously, we used the gene-breaking transposon to create 1200 transgenic zebrafish strains tagging protein-coding genes (Ichino et al., 2020), including the lrpprc locus. Here, we present and characterize a new genetic revertible animal model that recapitulates components of Leigh Syndrome French Canadian Type (LSFC), a mitochondrial disorder that includes diagnostic liver dysfunction. LSFC is caused by allelic variations in the LRPPRC gene, involved in mitochondrial mRNA polyadenylation and translation. lrpprc zebrafish homozygous mutants displayed biochemical and mitochondrial phenotypes similar to clinical manifestations observed in patients, including dysfunction in lipid homeostasis. We were able to rescue these phenotypes in the disease model using a liver-specific genetic model therapy, functionally demonstrating a previously under-recognized critical role for the liver in the pathophysiology of this disease.

A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene.

Previously we showed the generation of a protein trap library made with the gene-break transposon (GBT) in zebrafish (Danio rerio) that could be used to facilitate novel functional genome annotation towards understanding molecular underpinnings of human diseases (Ichino et al, 2020). Here, we report a significant application of this library for discovering essential genes for heart rhythm disorders such as sick sinus syndrome (SSS). SSS is a group of heart rhythm disorders caused by malfunction of the sinus node, the heart's primary pacemaker. Partially owing to its aging-associated phenotypic manifestation and low expressivity, molecular mechanisms of SSS remain difficult to decipher. From 609 GBT lines screened, we generated a collection of 35 zebrafish insertional cardiac (ZIC) mutants in which each mutant traps a gene with cardiac expression. We further employed electrocardiographic measurements to screen these 35 ZIC lines and identified three GBT mutants with SSS-like phenotypes. More detailed functional studies on one of the arrhythmogenic mutants, GBT411, in both zebrafish and mouse models unveiled Dnajb6 as a novel SSS causative gene with a unique expression pattern within the subpopulation of sinus node pacemaker cells that partially overlaps with the expression of hyperpolarization activated cyclic nucleotide gated channel 4 (HCN4), supporting heterogeneity of the cardiac pacemaker cells.

TMT-based quantitative proteomics analysis reveals the role of Notch signaling in FAdV-4-infected LMH cell.

Fowl adenovirus serotype 4 (FAdV-4) is recognized as a pathogen that causes hydropericardium syndrome. Irrespective of the pathway used by the virus to invade the chicken, the pathological characteristics of the disease include degeneration and necrosis of hepatocytes, formation of intranuclear inclusions, as well as inflammatory cell infiltration. Liver dysfunction constitutes one of the critical factors leading to death. Therefore, it is vital to investigate the virus-mediated severe pathological liver damage to further understand the pathogenesis of FAdV-4. Here, proteomics, a tandem mass tag (TMT)-based approach to directly analyze protein expression, was used to determine the protein expression during FAdV-4 proliferation in leghorn male hepatoma (LMH) cells. We identified 177 differentially expressed proteins associated with various biological processes and pathways. The functional enrichment analysis revealed that FAdV-4 could downregulate some signaling pathways in LMH cells, including NOD-like receptor signaling, RIG-I-like receptor signaling, NF-κB signaling, TNF signaling pathway, and Notch signaling, FoxO signaling, PI3K-Akt signaling, and autophagy. The results of proteomics screening suggested an association between FAdV-4 infection and Notch signaling in LMH in vitro, indicating that Notch signaling regulated the expression of inflammatory cytokines and interferons but not viral replication in LMH cells. These data contributed to the understanding of the immunopathogenesis and inflammopathogenesis of FAdV-4 infection and also provided valuable information for the further analysis of the molecular mechanisms underlying viral pathogenesis.

Genetic Susceptibility and Mechanisms Underlying the Pathogenesis of Anthracycline-Associated Cardiotoxicity.

Anthracyclines are chemotherapeutic agents widely used to treat a variety of cancers, and these drugs have revolutionized our management of cancer patients. The dose-dependent cardiotoxicity of anthracyclines, however, remains one of the leading causes of chemotherapy treatment-associated mortality in cancer survivors. Patient threshold doses leading to anthracycline-induced cardiotoxicity (AIC) are highly variable among affected patients. This variability is largely ascribed to genetic variants in individuals' genomes. Here, we briefly discuss the prevailing mechanisms underlying the pathogenesis of AIC, and then, we review the genetic variants, mostly identified through human genetic approaches and identified in cancer survivors. The identification of all genetic susceptibilities and elucidation of underlying mechanisms of AIC can help improve upfront risk prediction assessment for potentially severe cardiotoxicity disease and provide valuable insights into the understanding of AIC pathophysiology, which can be further leveraged to develop targeted pharmacogenetic therapies for those at high risk.

A Primer Genetic Toolkit for Exploring Mitochondrial Biology and Disease Using Zebrafish.

Mitochondria are a dynamic eukaryotic innovation that play diverse roles in biology and disease. The mitochondrial genome is remarkably conserved in all vertebrates, encoding the same 37-gene set and overall genomic structure, ranging from 16,596 base pairs (bp) in the teleost zebrafish (Danio rerio) to 16,569 bp in humans. Mitochondrial disorders are amongst the most prevalent inherited diseases, affecting roughly 1 in every 5000 individuals. Currently, few effective treatments exist for those with mitochondrial ailments, representing a major unmet patient need. Mitochondrial dysfunction is also a common component of a wide variety of other human illnesses, ranging from neurodegenerative disorders such as Huntington's disease and Parkinson's disease to autoimmune illnesses such as multiple sclerosis and rheumatoid arthritis. The electron transport chain (ETC) component of mitochondria is critical for mitochondrial biology and defects can lead to many mitochondrial disease symptoms. Here, we present a publicly available collection of genetic mutants created in highly conserved, nuclear-encoded mitochondrial genes in Danio rerio. The zebrafish system represents a potentially powerful new opportunity for the study of mitochondrial biology and disease due to the large number of orthologous genes shared with humans and the many advanced features of this model system, from genetics to imaging. This collection includes 15 mutant lines in 13 different genes created through locus-specific gene editing to induce frameshift or splice acceptor mutations, leading to predicted protein truncation during translation. Additionally, included are 11 lines created by the random insertion of the gene-breaking transposon (GBT) protein trap cassette. All these targeted mutant alleles truncate conserved domains of genes critical to the proper function of the ETC or genes that have been implicated in human mitochondrial disease. This collection is designed to accelerate the use of zebrafish to study many different aspects of mitochondrial function to widen our understanding of their role in biology and human disease.

An EGFP Knock-in Zebrafish Experimental Model Used in Evaluation of the Amantadine Drug Safety During Early Cardiogenesis.

Background: Drug exposure during gestation or in prematurely born children represents a significant risk to congenital heart disease (CHD). Amantadine is an antiviral agent also effective in the treatment of Parkinson's disease. However, while its potential side effects associated with tetralogy of fallot (ToF) and birth defects were implicated, its underlying etiologic mechanisms of action remain unknown. Here, we report teratogenic effects of amantadine drug during early cardiogenesis through developing a novel zebrafish (Danio rerio) knock-in (KI) animal model and explore the underlying mechanisms. Methods: Homologous recombination (HR) pathway triggered by CRISPR/Cas9 system was utilized to generate an enhanced green fluorescent protein (EGFP) KI zebrafish animal model. Dynamic fluorescence imaging coupled with a whole-mount in-situ hybridization (WISH) assay was employed to compare the spatial and temporal expression patterns of the EGFP reporter in the KI animal model with the KI-targeted endogenous gene. Heart morphology and EGFP expression dynamics in the KI animal models were monitored to assess cardiac side effects of different doses of amantadine hydrochloride. Expression of key genes required for myocardium differentiation and left-right (LR) asymmetry was analyzed using WISH and quantitative reverse transcription-PCR (RT-PCR). Results: A novel EGFP KI line targeted at the ventricular myosin heavy chain (vmhc) gene locus was successfully generated, in which EGFP reporter could faithfully recapitulate the endogenous expression dynamics of the ventricle chamber-specific expression of the vmhc gene. Amantadine drug treatment-induced ectopic expression of vmhc gene in the atrium and caused cardiac-looping or LR asymmetry defects to dose-dependently during early cardiogenesis, concomitant with dramatically reduced expression levels of key genes required for myocardium differentiation and LR asymmetry. Conclusion: We generated a novel zebrafish KI animal model in which EGFP reports the ventricle chamber-specific expression of vmhc gene dynamics that is useful to effectively assess drug safety on the cardiac morphology in vivo. Specifically, this study identified teratogenic effects of amantadine drug during early cardiogenesis dose dependent, which could be likely conveyed by inhibiting expression of key genes required for cardiac myocardium differentiation and LR asymmetry.

FAdV-4 induce autophagy via the endoplasmic reticulum stress-related unfolded protein response.

Hydropericardium syndrome caused by the fowl adenovirus serotype 4 (FAdV-4) is prevalent disease in China with a high mortality rate. Many studies have demonstrated some viral infections to induce stress in the endoplasmic reticulum (ER). When the ER stress exceeds or persists, it activates autophagy, eventually triggering the onset of diseases. However, no report has ever stated FAdV-4 infection to induce ER stress-mediated autophagy. Previous studies have identified FAdV-4 infection in triggering autophagy in the hepatocytes; however, the underlying mechanism of this induction remains unknown. This study investigated the mechanism of ER stress-mediated autophagy induced by FAdV-4 infection. Here, ER stress was found to be triggered by FAdV-4 infection, as evident from the increased expression of the ER stress marker glucose-regulated protein 78, and the dilated morphology of the ER. Three pathways linked with the unfolded protein response (UPR) were found to be triggered in the hepatocellular carcinoma cell line, which included the PKR-like ER protein kinase (PERK), transcription factor 6, and inositol-requiring enzyme 1 (IRE1) pathways, respectively. Additionally, our results demonstrated that autophagy is involved in the PERK-eukaryotic initiation factor 2 subunit - C/EBP homologous protein and IRE1-c-Jun-N-terminal kinase pathways. Furthermore, treatment with the small interfering RNAs, or specific chemical inhibitors for the two pathways were found to reduce the interfering activity and could suppress the FAdV-4 replication. Collectively, these results developed new insight into the mechanisms of FAdV-4-induced autophagy by activating the ER stress-related UPR pathway and provided the experimental bases and novel ideas for developing antiviral drugs.

A novel wireless ECG system for prolonged monitoring of multiple zebrafish for heart disease and drug screening studies.

Zebrafish and their mutant lines have been extensively used in cardiovascular studies. In the current study, the novel system, Zebra II, is presented for prolonged electrocardiogram (ECG) acquisition and analysis for multiple zebrafish within controllable working environments. The Zebra II is composed of a perfusion system, apparatuses, sensors, and an in-house electronic system. First, the Zebra II is validated in comparison with a benchmark system, namely iWORX, through various experiments. The validation displayed comparable results in terms of data quality and ECG changes in response to drug treatment. The effects of anesthetic drugs and temperature variation on zebrafish ECG were subsequently investigated in experiments that need real-time data assessment. The Zebra II's capability of continuous anesthetic administration enabled prolonged ECG acquisition up to 1 h compared to that of 5 min in existing systems. The novel, cloud-based, automated analysis with data obtained from four fish further provided a useful solution for combinatorial experiments and helped save significant time and effort. The system showed robust ECG acquisition and analytics for various applications including arrhythmia in sodium induced sinus arrest, temperature-induced heart rate variation, and drug-induced arrhythmia in Tg(SCN5A-D1275N) mutant and wildtype fish. The multiple channel acquisition also enabled the implementation of randomized controlled trials on zebrafish models. The developed ECG system holds promise and solves current drawbacks in order to greatly accelerate drug screening applications and other cardiovascular studies using zebrafish.

Inhibition of mTOR or MAPK ameliorates vmhcl/myh7 cardiomyopathy in zebrafish.

Myosin heavy chain 7 (MYH7) is a major causative gene for hypertrophic cardiomyopathy, but the affected signaling pathways and therapeutics remain elusive. In this research, we identified ventricle myosin heavy chain like (vmhcl) as a zebrafish homolog of human MYH7, and we generated vmhcl frameshift mutants. We noted vmhcl-based embryonic cardiac dysfunction (VEC) in the vmhcl homozygous mutants and vmhcl-based adult cardiomyopathy (VAC) phenotypes in the vmhcl heterozygous mutants. Using the VEC model, we assessed 7 known cardiomyopathy signaling pathways pharmacologically and 11 candidate genes genetically via CRISPR/Cas9 genome editing technology based on microhomology-mediated end joining (MMEJ). Both studies converged on therapeutic benefits of mTOR or mitogen-activated protein kinase (MAPK) inhibition of VEC. While mTOR inhibition rescued the enlarged nuclear size of cardiomyocytes, MAPK inhibition restored the prolonged cell shape in the VEC model. The therapeutic effects of mTOR and MAPK inhibition were later validated in the VAC model. Together, vmhcl/myh7 loss of function is sufficient to induce cardiomyopathy in zebrafish. The VEC and VAC models in zebrafish are amenable to both efficient genetic and chemical genetic tools, offering a rapid in vivo platform for discovering candidate signaling pathways of MYH7 cardiomyopathy.

Disruption of MAP7D1 Gene Function Increases the Risk of Doxorubicin-Induced Cardiomyopathy and Heart Failure.

Doxorubicin is a cornerstone chemotherapeutic drug widely used to treat various cancers; its dose-dependent cardiomyopathy, however, is one of the leading causes of treatment-associated mortality in cancer survivors. Patients' threshold doses leading to doxorubicin-induced cardiomyopathy (DIC) and heart failure are highly variable, mostly due to genetic variations in individuals' genomes. However, genetic susceptibility to DIC remains largely unidentified. Here, we combined a genetic approach in the zebrafish (Danio rerio) animal model with a genome-wide association study (GWAS) in humans to identify genetic susceptibility to DIC and heart failure. We firstly reported the cardiac and skeletal muscle-specific expression and sarcomeric localization of the microtubule-associated protein 7 domain-containing protein 1b (Map7d1b) in zebrafish, followed by expression validation in mice. We then revealed that disruption of the map7d1b gene function exaggerated DIC effects in adult zebrafish. Mechanistically, the exacerbated DIC are likely conveyed by impaired autophagic degradation and elevated protein aggregation. Lastly, we identified 2 MAP7D1 gene variants associated with cardiac functional decline and heart failure in cancer patients who received doxorubicin therapy. Together, this study identifies MAP7D1 as a clinically relevant susceptibility gene to DIC and heart failure, providing useful information to stratify cancer patients with a high risk of incurring severe cardiomyopathy and heart failure after receiving chemotherapy.

TFEB Overexpression, Not mTOR Inhibition, Ameliorates RagCS75Y Cardiomyopathy.

A de novo missense variant in Rag GTPase protein C (RagCS75Y) was recently identified in a syndromic dilated cardiomyopathy (DCM) patient. However, its pathogenicity and the related therapeutic strategy remain unclear. We generated a zebrafish RragcS56Y (corresponding to human RagCS75Y) knock-in (KI) line via TALEN technology. The KI fish manifested cardiomyopathy-like phenotypes and poor survival. Overexpression of RagCS75Y via adenovirus infection also led to increased cell size and fetal gene reprogramming in neonatal rat ventricle cardiomyocytes (NRVCMs), indicating a conserved mechanism. Further characterization identified aberrant mammalian target of rapamycin complex 1 (mTORC1) and transcription factor EB (TFEB) signaling, as well as metabolic abnormalities including dysregulated autophagy. However, mTOR inhibition failed to ameliorate cardiac phenotypes in the RagCS75Y cardiomyopathy models, concomitant with a failure to promote TFEB nuclear translocation. This observation was at least partially explained by increased and mTOR-independent physical interaction between RagCS75Y and TFEB in the cytosol. Importantly, TFEB overexpression resulted in more nuclear TFEB and rescued cardiomyopathy phenotypes. These findings suggest that S75Y is a pathogenic gain-of-function mutation in RagC that leads to cardiomyopathy. A primary pathological step of RagCS75Y cardiomyopathy is defective mTOR-TFEB signaling, which can be corrected by TFEB overexpression, but not mTOR inhibition.

The amino acid variants in HLA II molecules explain the major association with adult-onset Still's disease in the Han Chinese population.

Adult-onset Still's disease (AOSD) is a rare autoinflammatory disease with systemic involvement, and its pathophysiology remains unclear. Genome-wide association studies (GWAS) in the Chinese population have revealed an association between AOSD and the major histocompatibility complex (MHC) locus; however, causal variants in the MHC remain undetermined. In the present study, we identified independent amino-acid polymorphisms in human leukocyte antigen (HLA) molecules that are associated with Han Chinese patients with AOSD by fine-mapping the MHC locus. Through conditional analyses, we identified position 34 in HLA-DQα1 (p = 1.44 × 10-14) and Asn in HLA-DRß1 position 37 (p = 5.12 × 10-11) as the major determinants for AOSD. Moreover, we identified the associations for three main HLA class II alleles: HLA-DQB1*06:02 (OR = 2.70, p = 3.02 × 10-14), HLA-DRB1*15:01 (OR = 2.44, p = 3.66 × 10-13), and HLA-DQA1*01:02 (OR = 1.97, p = 1.09 × 10-9). This study reveals the relationship between functional variations in the class II HLA region and AOSD, implicating the MHC locus in the pathogenesis of AOSD.