LXRα Promotes Abdominal Aortic Aneurysm Formation Through UHRF1 Epigenetic Modification of miR-26b-3p.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a severe aortic disease without effective pharmacological approaches. The nuclear hormone receptor LXRα (liver X receptor α), encoded by the NR1H3 gene, serves as a critical transcriptional mediator linked to several vascular pathologies, but its role in AAA remains elusive. METHODS: Through integrated analyses of human and murine AAA gene expression microarray data sets, we identified NR1H3 as a candidate gene regulating AAA formation. To investigate the role of LXRα in AAA formation, we used global Nr1h3-knockout and vascular smooth muscle cell-specific Nr1h3-knockout mice in 2 AAA mouse models induced with angiotensin II (1000 ng·kg·min; 28 days) or calcium chloride (CaCl2; 0.5 mol/L; 42 days). RESULTS: Upregulated LXRα was observed in the aortas of patients with AAA and in angiotensin II- or CaCl2-treated mice. Global or vascular smooth muscle cell-specific Nr1h3 knockout inhibited AAA formation in 2 mouse models. Loss of LXRα function prevented extracellular matrix degeneration, inflammation, and vascular smooth muscle cell phenotypic switching. Uhrf1, an epigenetic master regulator, was identified as a direct target gene of LXRα by integrated analysis of transcriptome sequencing and chromatin immunoprecipitation sequencing. Susceptibility to AAA development was consistently enhanced by UHRF1 (ubiquitin-like containing PHD and RING finger domains 1) in both angiotensin II- and CaCl2-induced mouse models. We then determined the CpG methylation status and promoter accessibility of UHRF1-mediated genes using CUT&Tag (cleavage under targets and tagmentation), RRBS (reduced representation bisulfite sequencing), and ATAC-seq (assay for transposase-accessible chromatin with sequencing) in vascular smooth muscle cells, which revealed that the recruitment of UHRF1 to the promoter of miR-26b led to DNA hypermethylation accompanied by relatively closed chromatin states, and caused downregulation of miR-26b expression in AAA. Regarding clinical significance, we found that underexpression of miR-26b-3p correlated with high risk in patients with AAA. Maintaining miR-26b-3p expression prevented AAA progression and alleviated the overall pathological process. CONCLUSIONS: Our study reveals a pivotal role of the LXRα/UHRF1/miR-26b-3p axis in AAA and provides potential biomarkers and therapeutic targets for AAA.

Characterizing the cellular and molecular variabilities of peripheral immune cells in healthy recipients of BBIBP-CorV inactivated SARS-CoV-2 vaccine by single-cell RNA sequencing.

Over 3 billion doses of inactivated vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been administered globally. However, our understanding of the immune cell functional transcription and T cell receptor (TCR)/B cell receptor (BCR) repertoire dynamics following inactivated SARS-CoV-2 vaccination remains poorly understood. Here, we performed single-cell RNA and TCR/BCR sequencing on peripheral blood mononuclear cells at four time points after immunization with the inactivated SARS-CoV-2 vaccine BBIBP-CorV. Our analysis revealed an enrichment of monocytes, central memory CD4+ T cells, type 2 helper T cells and memory B cells following vaccination. Single-cell TCR-seq and RNA-seq comminating analysis identified a clonal expansion of CD4+ T cells (but not CD8+ T cells) following a booster vaccination that corresponded to a decrease in the TCR diversity of central memory CD4+ T cells and type 2 helper T cells. Importantly, these TCR repertoire changes and CD4+ T cell differentiation were correlated with the biased VJ gene usage of BCR and the antibody-producing function of B cells post-vaccination. Finally, we compared the functional transcription and repertoire dynamics in immune cells elicited by vaccination and SARS-CoV-2 infection to explore the immune responses under different stimuli. Our data provide novel molecular and cellular evidence for the CD4+ T cell-dependent antibody response induced by inactivated vaccine BBIBP-CorV. This information is urgently needed to develop new prevention and control strategies for SARS-CoV-2 infection. (ClinicalTrials.gov Identifier: NCT04871932).

A natural DNMT1 mutation elevates the fetal hemoglobin level via epigenetic derepression of the γ-globin gene in ß-thalassemia.

DNA methyltransferase 1 (DNMT1) is a major epigenetic regulator of the formation of large macromolecular complexes that repress human γ-globin expression by maintaining DNA methylation. However, very little is known about the association of DNMT1 variants with ß-thalassemia phenotypes. We systematically investigated associations between variants in DNMT1 and phenotypes in 1142 ß-thalassemia subjects and identified a novel missense mutation (c.2633G>A, S878F) in the DNMT1 bromo-adjacent homology-1 (BAH1) domain. We functionally characterized this mutation in CD34+ cells from patients and engineered HuDEP-2 mutant cells. Our results demonstrate that DNMT1 phosphorylation is abrogated by substituting serine with phenylalanine at position 878, resulting in lower stability and catalytic activity loss. S878F mutation also attenuated DNMT1 interactions with BCL11A, GATA1, and HDAC1/2, and reduced recruitment of DNMT1 to the γ-globin (HBG) promoters, leading to epigenetic derepression of γ-globin expression. By analyzing the F-cell pattern, we demonstrated that the effect of DNMT1 mutation on increased fetal hemoglobin (HbF) is heterocellular. Furthermore, introduction of S878F mutation into erythroid cells by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) recapitulated γ-globin reactivation. Thus, the natural S878F DNMT1 mutation is a novel modulator of HbF synthesis and represents a potential new therapeutic target for ß-hemoglobinopathies.

Characterization of a Novel 71.8 kb α0-Thalassemia Deletion and Subsequent Summary of a Practical Procedure for Thalassemia Molecular Diagnosis.

Thalassemia is the most common monogenic disorder around the world. Based on the principle of genotype-phenotype correlation, identification of thalassemia mutations is the essential prerequisite for clinical diagnosis and management. Because only common mutations are routinely detected, the identification of rare or undetermined mutations is a challenge for clinical laboratories. Herein, a proband presenting with inconsistent phenotype-genotype correlation after routine molecular screening was investigated by multiplex ligation-dependent probe amplification (MLPA), targeted-next generation sequencing (targeted-NGS), gap-polymerase chain reaction (gap-PCR) and Sanger sequencing. Eventually, a novel 71.8 kb deletion (- -71.8) was identified and characterized, which included HBZ (ζ), HBA2 (α2), and HBA1 (α1) genes and was causing α0-thalassemia (α0-thal). Furthermore, we summarized a practical procedure based on accumulated experience in studies and clinical practice, which can be a guide for molecular screening and clinical diagnosis of thalassemia, especially for identification of undetermined or novel mutations.

Putative regulators for the continuum of erythroid differentiation revealed by single-cell transcriptome of human BM and UCB cells.

Fine-resolution differentiation trajectories of adult human hematopoietic stem cells (HSCs) involved in the generation of red cells is critical for understanding dynamic developmental changes that accompany human erythropoiesis. Using single-cell RNA sequencing (scRNA-seq) of primary human terminal erythroid cells (CD34-CD235a+) isolated directly from adult bone marrow (BM) and umbilical cord blood (UCB), we documented the transcriptome of terminally differentiated human erythroblasts at unprecedented resolution. The insights enabled us to distinguish polychromatic erythroblasts (PolyEs) at the early and late stages of development as well as the different development stages of orthochromatic erythroblasts (OrthoEs). We further identified a set of putative regulators of terminal erythroid differentiation and functionally validated three of the identified genes, AKAP8L, TERF2IP, and RNF10, by monitoring cell differentiation and apoptosis. We documented that knockdown of AKAP8L suppressed the commitment of HSCs to erythroid lineage and cell proliferation and delayed differentiation of colony-forming unit-erythroid (CFU-E) to the proerythroblast stage (ProE). In contrast, the knockdown of TERF2IP and RNF10 delayed differentiation of PolyE to OrthoE stage. Taken together, the convergence and divergence of the transcriptional continuums at single-cell resolution underscore the transcriptional regulatory networks that underlie human fetal and adult terminal erythroid differentiation.

Use of SuperCT for Enhanced Characterization of Single-Cell Transcriptomic Profiles.

Characterizing the cell identity in a heterogeneous tissue is essential to the in-depth understanding of this sample. Existing single-cell techniques (e.g., flow cytometry or in situ cell florescent imaging) allow us to do so using the high/low signal of a combination of multiple signature molecules or even of a single marker. Recent advance of single-cell RNA-seq technology profiles the entire transcriptome of individual cells. Using a few marker genes to characterize cell type in this new technique is less reliable due to the high noise level and the dynamic transcription behavior. Nonetheless, the "noisy" but high-throughput transcriptome profiles provide adequate information to reveal the cellular identity and to understand the detail of the molecular characteristics. In this chapter, we will demonstrate a new method that is based on the supervised learning of the single-cell transcriptome profiles of many different known cell types. We will demonstrate how this technique solves the cellular identity problem.

LOVD-DASH: A comprehensive LOVD database coupled with diagnosis and an at-risk assessment system for hemoglobinopathies.

Hemoglobinopathies are the most common monogenic disorders worldwide. Substantial effort has been made to establish databases to record complete mutation spectra causing or modifying this group of diseases. We present a variant database which couples an online auxiliary diagnosis and at-risk assessment system for hemoglobinopathies (DASH). The database was integrated into the Leiden Open Variation Database (LOVD), in which we included all reported variants focusing on a Chinese population by literature peer review-curation and existing databases, such as HbVar and IthaGenes. In addition, comprehensive mutation data generated by high-throughput sequencing of 2,087 hemoglobinopathy patients and 20,222 general individuals from southern China were also incorporated into the database. These sequencing data enabled us to observe disease-causing and modifier variants responsible for hemoglobinopathies in bulk. Currently, 371 unique variants have been recorded; 265 of 371 were described as disease-causing variants, whereas 106 were defined as modifier variants, including 34 functional variants identified by a quantitative trait association study of this high-throughput sequencing data. Due to the availability of a comprehensive phenotype-genotype data set, DASH has been established to automatically provide accurate suggestions on diagnosis and genetic counseling of hemoglobinopathies. LOVD-DASH will inspire us to deal with clinical genotyping and molecular screening for other Mendelian disorders.

Mutations in C1orf194, encoding a calcium regulator, cause dominant Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease is a hereditary motor and sensory neuropathy exhibiting great clinical and genetic heterogeneity. Here, the identification of two heterozygous missense mutations in the C1orf194 gene at 1p21.2-p13.2 with Charcot-Marie-Tooth disease are reported. Specifically, the p.I122N mutation was the cause of an intermediate form of Charcot-Marie-Tooth disease, and the p.K28I missense mutation predominately led to the demyelinating form. Functional studies demonstrated that the p.K28I variant significantly reduced expression of the protein, but the p.I122N variant increased. In addition, the p.I122N mutant protein exhibited the aggregation in neuroblastoma cell lines and the patient's peroneal nerve. Either gain-of-function or partial loss-of-function mutations to C1ORF194 can specify different causal mechanisms responsible for Charcot-Marie-Tooth disease with a wide range of clinical severity. Moreover, a knock-in mouse model confirmed that the C1orf194 missense mutation p.I121N led to impairments in motor and neuromuscular functions, and aberrant myelination and axonal phenotypes. The loss of normal C1ORF194 protein altered intracellular Ca2+ homeostasis and upregulated Ca2+ handling regulatory proteins. These findings describe a novel protein with vital functions in peripheral nervous systems and broaden the causes of Charcot-Marie-Tooth disease, which open new avenues for the diagnosis and treatment of related neuropathies.

Thalassaemia intermedia caused by coinheritance of a ß-thalassaemia mutation and a de novo duplication of α-globin genes in the paternal allele.

Next generation sequencing identified a de novo, 204 kb, tandem duplication (αααα204 ) in the α-globin gene cluster of a Chinese thalassaemia intermedia patient. Haplotype analysis showed that the duplicated chromosome was of paternal origin. Molecular analysis of genomic DNA from the patient's lymphocytes, hair follicles, buccal mucosa cells, his father's lymphocytes and sperm cells excluded the possibility of somatic or germinal mosaicism. The analysis also indicated that this duplication arose during spermatogenesis. The microhomology in the breakpoint was found and suggested that this duplication could be formed by a coupled homologous and non-homologous recombination mechanism.

A novel 223 kb deletion in the beta-globin gene cluster was identified in a Chinese thalassemia major patient.

INTRODUCTION: Although mutations in the human beta-globin gene cluster are essentially point mutations, several large deletions have been described in recent years. METHODS: We have identified a novel 223 kb deletion in a Chinese patient by multiplex ligation-dependent probe amplification and characterized it by next-generation sequencing, Gap-PCR, and DNA sequence analysis. RESULTS: The deletion extends from the 3'UTR of the δ globin gene (HBD) to 215 kb downstream of the HBB. Compound heterozygous with the typical ß-thalassemia-CD41-42(-CTTT) mutation, the proband presented with microcytosis and hypochromic red cells, and required regulate transfusion. The patient was clinically diagnosed with thalassemia major. CONCLUSION: Our study widens the mutation spectrum of ß-thalassemia. In addition, this case may spark future studies of the regulatory regions of the beta-globin gene cluster.

Analysis of variants in upstream open reading frames of human globin-related genes.

ß-thalassemia is an autosomal recessive monogenic disease that is caused by defects in the production of ß-like globin chains. Activation of γ-globin gene and the increase in fetal hemoglobin expression have been demonstrated as one of the most important factors to ameliorate the clinical outcome of ß-thalassemia patients. In this study, 202 genes or miRNAs associated with human hemoglobin gene expression from 1802 ß-thalassemia patients were analyzed with target capture and next generation sequencing strategies in terms of functional variants that might affect hemoglobin gene expression. The subsequent bioinformatics analysis included assessments of sequence quality, the variants within the target regions and the 5'UTR with potential effects on upstream open reading frames (uORFs). Among the 41 variants in 5'UTR potentially affecting the uORFs identified in the study, two variants (chr19: 41859418 G > A and chr1:153606541 C > T) were experimentally validated with dual-luciferase assays to be capable of significantly down-regulating the expression of TGFB1 and CHTOP gene, respectively. The present study demonstrated a system suitable for evaluating the importance of variants in 5'UTRs affecting uORFs in 202 human genes associated with hemoglobin expression. Research with this approach could provide potential targets that may contribute to the clinical phenotypes and provide biomarkers for precise diagnosis of ß-thalassemia.