陈文
中国医学科学院阜外医院 心血管疾病国家重点实验室
Ebstein's anomaly, a rare congenital heart disease, is distinguished by the failure of embryological delamination of the tricuspid valve leaflets from the underlying primitive right ventricle myocardium. Gaining insight into the genetic basis of Ebstein's anomaly allows a more precise definition of its pathogenesis. In this study, two distinct cohorts from the Chinese Han population were included: a case-control cohort consisting of 82 unrelated cases and 125 controls without cardiac phenotypes and a trio cohort comprising 36 parent-offspring trios. Whole-exome sequencing data from all 315 participants were utilized to identify qualifying variants, encompassing rare (minor allele frequency < 0.1% from East Asians in the gnomAD database) functional variants and high-confidence (HC) loss-of-function (LoF) variants. Various statistical models, including burden tests and variance-component models, were employed to identify rare variants, genes, and biological pathways associated with Ebstein's anomaly. Significant associations were noted between Ebstein's anomaly and rare HC LoF variants found in genes related to the matrisome, a collection of extracellular matrix (ECM) components. Specifically, 47 genes with HC LoF variants were exclusively or predominantly identified in cases, while nine genes showed such variants in the probands. Over half of unrelated cases (n = 42) and approximately one-third of probands (n = 12) were found to carry one or two LoF variants in these prioritized genes. These results highlight the role of the matrisome in the pathogenesis of Ebstein's anomaly, contributing to a better understanding of the genetic architecture underlying this condition. Our findings hold the potential to impact the genetic diagnosis and treatment approaches for Ebstein's anomaly.
HGG advances 2024
Hypertrophic cardiomyopathy (HCM) is the most common cardiac genetic disorder characterized by cardiomyocyte hypertrophy and cardiac fibrosis. Pathological cardiac remodeling in the myocardium of HCM patients may progress to heart failure. An in-depth elucidation of the lineage-specific changes in pathological cardiac remodeling of HCM is pivotal for the development of therapies to mitigate the progression. Here, we performed single-nucleus RNA-seq of the cardiac tissues from HCM patients or healthy donors and conducted spatial transcriptomic assays on tissue sections from patients. Unbiased clustering of 55,122 nuclei from HCM and healthy conditions revealed 9 cell lineages and 28 clusters. Lineage-specific changes in gene expression, subpopulation composition, and intercellular communication in HCM were discovered through comparative analyses. According to the results of pseudotime ordering, differential expression analysis, and differential regulatory network analysis, potential key genes during the transition towards a failing state of cardiomyocytes such as FGF12, IL31RA, and CREB5 were identified. Transcriptomic dynamics underlying cardiac fibroblast activation were also uncovered, and potential key genes involved in cardiac fibrosis were obtained such as AEBP1, RUNX1, MEOX1, LEF1, and NRXN3. Using the spatial transcriptomic data, spatial activity patterns of the candidate genes, pathways, and subpopulations were confirmed on patient tissue sections. Moreover, we showed experimental evidence that in vitro knockdown of AEBP1 could promote the activation of human cardiac fibroblasts, and overexpression of AEBP1 could attenuate the TGFβ-induced activation. Our study provided a comprehensive analysis of the lineage-specific regulatory changes in HCM, which laid the foundation for targeted drug development in HCM.
Cell discovery 2023
Ebstein's anomaly is a rare congenital heart disease characterized by tricuspid valve downward displacement and is associated with additional cardiac phenotypes such as left ventricle non-compaction. The genetic basis of Ebstein's anomaly has yet to be fully elucidated, although several genes (e.g., NKX2-5, MYH7, TPM1, and FLNA) may contribute to Ebstein's anomaly. Here, in two Ebstein's anomaly families (a three-generation family and a trio), we identified independent heterozygous nonsense variants in laminin subunit 3 α (LAMA3), cosegregated with phenotypes in families with reduced penetrance. Furthermore, knocking out Lama3 in mice revealed that haploinsufficiency of Lama3 led to Ebstein's malformation of the tricuspid valve and an abnormal basement membrane structure. In conclusion, we identified a novel gene-disease association of LAMA3 implicated in Ebstein's anomaly, and the findings extended our understanding of the role of the extracellular matrix in Ebstein's anomaly etiology.
HGG advances 2023
Congenital heart disease (CHD) is one of themost common causes of major birth defects, with a prevalence of 1%. Although an increasing number of studies have reported the etiology of CHD, the findings scattered throughout the literature are difficult to retrieve and utilize in research and clinical practice. We therefore developed CHDbase, an evidence-based knowledgebase of CHD-related genes and clinical manifestations manually curated from 1114 publications, linking 1124susceptibility genes and 3591 variations to more than 300 CHD types and related syndromes. Metadata such as the information of each publication and the selected population and samples, the strategy of studies, and the major findings of studies were integrated with each item of the research record. We also integrated functional annotations through parsing ∼ 50 databases/tools to facilitate the interpretation of these genes and variations in disease pathogenicity. We further prioritized the significance of these CHD-related genes with a gene interaction network approach and extracted a core CHD sub-network with 163 genes. The clear genetic landscape of CHD enables the phenotype classification based on the shared genetic origin. Overall, CHDbase provides a comprehensive and freely available resource to study CHD susceptibilities, supporting a wide range of users in the scientific and medical communities. CHDbase is accessible at http://chddb.fwgenetics.org.
Genomics, proteomics & bioinformatics 2023
Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening condition characterized by medial layer degeneration of the thoracic aorta. A thorough understanding of the regulator changes during pathogenesis is essential for medical therapy development. To delineate the cellular and molecular changes during the development of TAAD, we performed single-cell RNA sequencing of thoracic aortic cells from β-aminopropionitrile-induced TAAD mouse models at three time points that spanned from the early to the advanced stages of the disease. Comparative analyses were performed to delineate the temporal dynamics of changes in cellular composition, lineage-specific regulation, and cell-cell communications. Excessive activation of stress-responsive and Toll-like receptor signaling pathways contributed to the smooth muscle cell senescence at the early stage. Three subpopulations of aortic macrophages were identified, i.e., Lyve1+ resident-like, Cd74high antigen-presenting, and Il1rn+/Trem1+ pro-inflammatory macrophages. In both mice and humans, the pro-inflammatory macrophage subpopulation was found to represent the predominant source of most detrimental molecules. Suppression of macrophage accumulation in the aorta with Ki20227 could significantly decrease the incidence of TAAD and aortic rupture in mice. Targeting the Il1rn+/Trem1+ macrophage subpopulation via blockade of Trem1 using mLR12 could significantly decrease the aortic rupture rate in mice. We present the first comprehensive analysis of the cellular and molecular changes during the development of TAAD at single-cell resolution. Our results highlight the importance of anti-inflammation therapy in TAAD, and pinpoint the macrophage subpopulation as the predominant source of detrimental molecules for TAAD. Targeting the IL1RN+/TREM1+ macrophage subpopulation via blockade of TREM1 may represent a promising medical treatment.
Cell discovery 2022
Cancer-associated lymphedema frequently occurs following lymph node resection for cancer treatment. However, we still lack effective targeted medical therapies for the treatment or prevention of this complication. An in-depth elucidation of the cellular alterations in subcutaneous adipose tissues of lymphedema is essential for medical development. We performed single-cell RNA sequencing of 70,209 cells of the stromal vascular fraction of adipose tissues from lymphedema patients and healthy donors. Four subpopulations of adipose-derived stromal cells (ASCs) were identified. Among them, the PRG4+/CLEC3B+ ASC subpopulation c3 was significantly expanded in lymphedema and related to adipose tissue fibrosis. Knockdown of CLEC3B in vitro could significantly attenuate the fibrogenesis of ASCs from patients. Adipose tissues of lymphedema displayed a striking depletion of LYVE+ anti-inflammatory macrophages and exhibited a pro-inflammatory microenvironment. Pharmacological blockage of Trem1, an immune receptor predominantly expressed by the pro-inflammatory macrophages, using murine LR12, a dodecapeptide, could significantly alleviate lymphedema in a mouse tail model. Cell-cell communication analysis uncovered a perivascular ligand-receptor interaction module among ASCs, macrophages, and vascular endothelial cells. We provided a comprehensive analysis of the lineage-specific changes in the adipose tissues from lymphedema patients at a single-cell resolution. CLEC3B was found to be a potential target for alleviating adipose tissue fibrosis. Pharmacological blockage of TREM1 using LR12 could serve as a promising medical therapy for treating lymphedema.
Cell discovery 2022
Keloids are a benign dermal fibrotic disorder with features similar to malignant tumors. keloids remain a therapeutic challenge and lack medical therapies, which is partially due to the incomplete understanding of the pathogenesis mechanism. We performed single-cell RNA-sequencing of 28,064 cells from keloid skin tissue and adjacent relatively normal tissue. Unbiased clustering revealed substantial cellular heterogeneity of keloid tissue, which included 21 clusters assigned to 11 cell lineages. We observed significant expansion of fibroblast and vascular endothelial cell subpopulations in keloids, reflecting their strong association with keloid pathogenesis. Comparative analyses were performed to identify the dysregulated pathways, regulators and ligand-receptor interactions in keloid fibroblasts and vascular endothelial cells. Our results highlight the roles of TGFβ and Eph-ephrin signaling pathways in both the aberrant fibrogenesis and angiogenesis of keloids. Critical regulators probably involved in the fibrogenesis of keloid fibroblasts, such as TWIST1, FOXO3 and SMAD3, were identified. TWIST1 inhibitor harmine could significantly suppress the fibrogenesis of keloid fibroblasts. In addition, tumor-related pathways were activated in keloid fibroblasts and vascular endothelial cells, which may be responsible for the malignant features of keloids. Our study put insights into the pathogenesis of keloids and provides potential targets for medical therapies.
The Journal of investigative dermatology 2022
Anomalous pulmonary venous return (APVR) is a potentially lethal congenital heart disease. Elucidating the genetic etiology is crucial for understanding its pathogenesis and improving clinical practice, whereas its genetic basis remains largely unknown because of complex genetic etiology. We thus performed whole-exome sequencing for 144 APVR patients and 1636 healthy controls and report a comprehensive atlas of APVR-related rare genetic variants. Novel singleton, loss-of-function and deleterious missense variants (DVars) were enriched in patients, particularly for genes highly expressed in the developing human heart at the critical time point for pulmonary veins draining into the left atrium. Notably, PLXND1, encoding a receptor for semaphorins, represents a strong candidate gene of APVR (adjusted P = 1.1e-03, odds ratio: 10.9-69.3), accounting for 4.17% of APVR. We further validated this finding in an independent cohort consisting of 82 case-control pairs. In these two cohorts, eight DVars were identified in different patients, which convergently disrupt the GTPase-activating protein-related domain of PLXND1. All variant carriers displayed strikingly similar clinical features, in that all anomalous drainage of pulmonary vein(s) occurred on the right side and incorrectly connected to the right atrium, which may represent a novel subtype of APVR for molecular diagnosis. Studies in Plxnd1 knockout mice further revealed the effects of PLXND1 deficiency on severe heart and lung defects and cellular abnormalities related to APVR such as abnormal migration and vascular formation of vascular endothelial cells. These findings indicate the important role of PLXND1 in APVR pathogenesis, providing novel insights into the genetic etiology and molecular subtyping for APVR.
Human molecular genetics 2022
AIMS:Thoracic aortic dissection (TAD) is a life-threatening disease with no effective drug therapy thus far. New therapeutic targets and indications for timely surgical intervention are urgently needed. Our aim is to investigate new pathological mechanisms and potential biomarkers of TAD through global metabolomic profiling of aortic aneurysm and dissection patients.METHODS AND RESULTS:We performed untargeted metabolomics to determine plasma metabolite concentrations in an aortic disease cohort, including 70 thoracic aortic aneurysm (TAA) and 70 TAD patients, as well as 70 healthy controls. Comparative analysis revealed that sphingolipid, especially its core metabolite C18-ceramide, was significantly distinguished in TAD patients but not in TAA patients, which was confirmed by subsequent quantitative analysis of C18-ceramide in a validation cohort. By analyzing our existing multiomics data in aortic tissue in a murine TAD model and TAD patients, we found that an enhanced ceramide de novo synthesis pathway in macrophages might contribute to the elevated ceramide. Inhibition of the ceramide de novo synthesis pathway by myriocin markedly alleviated BAPN-induced aortic inflammation and dissection in mice. In vitro studies demonstrated that exogenous C18-ceramide promoted macrophage inflammation and matrix metalloprotein (MMP) expression through the NLRP3-caspase 1 pathway. In contrast, inhibition of endogenous ceramide synthesis by myriocin attenuated lipopolysaccharide (LPS)-induced macrophage inflammation.CONCLUSIONS:Our findings demonstrated that ceramide metabolism disturbance might play a vital role in TAD development by aggravating aortic inflammation through the NLRP3 pathway, possibly providing a new target for pharmacological therapy and a potential biomarker of TAD.
Frontiers in cardiovascular medicine 2022
OBJECTIVES:Congenitally corrected transposition of the great arteries (ccTGA) is a rare and complex congenital heart disease with the characteristics of double discordance. Enormous co-existed anomalies are the culprit of prognosis evaluation and clinical decision. We aim at delineating a novel ccTGA clustering modality under human phenotype ontology (HPO) instruction and elucidating the relationship between phenotypes and prognosis in patients with ccTGA.METHODS:A retrospective review of 270 patients diagnosed with ccTGA in Fuwai hospital from 2009 to 2020 and cross-sectional follow-up were performed. HPO-instructed clustering method was administered in ccTGA risk stratification. Kaplan-Meier survival, Landmark analysis, and cox regression analysis were used to investigate the difference of outcomes among clusters.RESULTS:The median follow-up time was 4.29 (2.07-7.37) years. A total of three distinct phenotypic clusters were obtained after HPO-instructed clustering with 21 in cluster 1, 136 in cluster 2, and 113 in cluster 3. Landmark analysis revealed significantly worse mid-term outcomes in all-cause mortality (p = 0.021) and composite endpoints (p = 0.004) of cluster 3 in comparison with cluster 1 and cluster 2. Multivariate analysis indicated that pulmonary arterial hypertension (PAH), atrioventricular septal defect (AVSD), and arrhythmia were risk factors for composite endpoints. Moreover, the surgical treatment was significantly different among the three groups (p < 0.001) and surgical strategies had different effects on the prognosis of the different phenotypic clusters.CONCLUSIONS:Human phenotype ontology-instructed clustering can be a potentially powerful tool for phenotypic risk stratification in patients with complex congenital heart diseases, which may improve prognosis prediction and clinical decision.
Frontiers in cardiovascular medicine 2021
