Ke Hao

Fetal alcohol spectrum disorders (FASD) exhibit wide phenotypic variability not explained by prenatal alcohol exposure (PAE) alone. While genetic factors may modify vulnerability, there are no prior FASD genome-wide association studies (GWAS). We performed genome-wide single-marker and gene-based analyses of two South African prospective birth cohorts (Cape Town Longitudinal Cohorts (CTLC); n=328 mother-child dyads) and the US Collaborative Initiative on Fetal Alcohol Spectrum Disorders cohort (CIFASD n=429 children only). We examined main genetic and gene-by-PAE interaction effects on working memory, recognition memory, height, and FASD diagnosis using single-marker and gene-based analyses and the Haplotype-based Transmission Disequilibrium Test (HTDT), which integrates maternal-child genetics. Genome-wide genotype array data were used; TOPMed-imputed data were used to refine results. Risk-modifying genes were then tested for differential mRNA expression based on PAE in placenta and child blood. Gene*PAE interaction gene-based analyses identified STX6 associated with height-for-age Z-scores (p=2.2×10⁻⁶) and FOXD3 with recognition memory performance (p=4.7×10⁻⁶) in children. Single-marker analysis detected one child SNP approaching significance (rs2833924, p=8.8x10-8) with Gene*PAE interaction for working memory performance. CTLC+CIFASD meta-analysis in children identified one significant locus (HTR1E; pmeta=2.75×10⁻⁶) with Gene*PAE interaction for working memory performance. The HTDT test in CTLC imputed data identified three haplotypes associated with FASD diagnosis risk: COP1 (padj=0.029), WWOX (padj=2×10⁻4), TMEM38B (padj= 0.019). WWOX, STX6, and TMEM38B were differentially expressed in placenta and/or child blood based on PAE. In this first-ever genome-wide investigation of maternal and child genetics in FASD, we identified multiple biologically plausible risk-modifying loci for FASD neurobehavioral, growth, and FASD diagnosis outcomes.

Background and Aims: Clonal haematopoiesis of indeterminate potential (CHIP) has been associated with cardiovascular risk, but its prognostic relevance and mechanistic role in coronary artery disease (CAD) remains incompletely understood. This study investigated the association between CHIP and all-cause mortality in CAD and explored the cellular and molecular mechanisms, focusing on TET2 mutations. Methods: Targeted deep sequencing of 13 CHIP driver genes in 8612 patients with angiographically confirmed CAD was performed. Clonal haematopoiesis of indeterminate potential carriers (variant allele frequency ≥2%) were propensity-score matched 1:1 to non-carriers. Mortality was assessed over 3 years. Mechanistic insights were derived from post-mortem high-sensitivity plaque proteomics (MISSION), RNA sequencing from carotid plaques (Athero-Express), monocyte-derived macrophage transcriptomes (STARNET), and CRISPR/Cas9-generated TET2+/− macrophages in vitro. Results: Clonal haematopoiesis of indeterminate potential was associated with increased 3-year mortality (hazard ratio 1.39, 95% confidence interval 1.16–1.65, P < .001) in 2389 matched pairs. Mutations in TET2, ASXL1, DNMT3A, JAK2, PPM1D, SF3B1, SRSF2, and U2AF1 individually conferred higher mortality risk. In human plaques, CHIP mutations were found in lesional macrophages. TET2 CHIP carriers showed increased necrotic core size, inflammation, and reduced plaque stability. Multi-omics profiling revealed up-regulation of lipid metabolism and inflammatory pathways. TET2+/− macrophages exhibited increased LDLR expression and lipid uptake, linked to enhanced chromatin accessibility at the LDLR promoter. These findings were confirmed in carotid plaques, which showed increased LDLR and inflammasome-related gene expression in TET2 CHIP carriers. Conclusions: Clonal haematopoiesis of indeterminate potential is a predictor of mortality in CAD patients. TET2 mutations promote a pro-atherogenic macrophage phenotype via LDLR up-regulation and inflammatory activation, linking epigenetic dysregulation to adverse outcomes in CAD.

Background and Aims: Translating human genomic discoveries into mechanistic insights requires linking genetic variations to candidate genes and their causal functional phenotypes. Genome-wide association studies have consistently identified LIPA (lipase A, lysosomal acid type) as a risk locus for coronary artery disease, with previous analyses prioritising LIPA as a likely causal gene. However, functional studies elucidating causal variants, regulatory mechanisms, target cell types, and their causal impact on atherosclerosis have been lacking. This study aims to address this gap by establishing the variant-to-function relationship at the LIPA locus. Methods: Post-genome-wide association study pipelines and molecular biology techniques, including expression quantitative trait loci analysis, Tri-HiC, luciferase assay, CRISPRi, allele-specific binding, motif analysis, and electrophoretic mobility shift assay, were used to link functional variants to target genes and define the direction of their regulatory effects in causal cell types. To determine how increased myeloid LIPA impacts atherosclerosis, myeloid-specific Lipa overexpression mice on an Ldlr−/− background were generated. Results: Coronary artery disease–risk alleles in the LIPA locus increase LIPA expression and enzyme activity specifically in monocytes/macrophages by enhancing PU.1 binding to an intronic enhancer region that interacts with the LIPA promoter. Myeloid-specific Lipa overexpression in Ldlr−/− mice fed a western diet resulted in larger atherosclerotic lesions, accompanied by altered macrophage function, characterized by increased accumulation of lesional macrophages derived from circulating monocytes, reduced neutral lipid content, and up-regulation of integrin and extracellular matrix pathway genes. Conclusions: The work establishes a direct causal link between LIPA-risk alleles and increased monocyte/macrophage LIPA that exacerbates atherosclerosis, bridging human functional genomic evidence to the mechanistic understanding of coronary artery disease.

Carotid stenosis causes ischemic stroke in both sexes, but the clinical presentation and plaque characteristics differ. Here we run deep single-cell sequencing of 7,690 human carotid plaque cells from male and female patients. While we found no sex differences in major cell types, we identified a predominance of the osteogenic phenotype in smooth muscle cells, immunomodulating macrophages (MPs) and endothelial cells (ECs) undergoing endothelial-to-mesenchymal transition in females. In males, we found smooth muscle cells with the chondrocytic phenotype, MPs involved in tissue remodeling and ECs with angiogenic activity. Sex-biased subcellular clusters were integrated with tissue-specific gene-regulatory networks (GRNs) from the Stockholm–Tartu Atherosclerosis Reverse Network Engineering Task study. We identified GRN195 involved in angiogenesis and T cell-mediated cytotoxicity in male ECs, while in females, we found GRN33 and GRN122 related to TREM2⁻/TREM1⁺ MPs and endothelial-to-mesenchymal transition. The impact of GRN195 on EC proliferation in males was functionally validated, providing evidence for potential therapy targets for atherosclerosis that are sex specific.

Vehicle nitrogen oxides (NO_x) significantly increase nitrogen dioxide (NO₂) exposure in traffic-related environments. The NO₂/NO_x ratios are crucial for accurate NO₂ modeling and are closely linked to public health concerns. In 2020, we used a mobile platform to follow test trucks (plume-chasing) that were installed with a portable emission measuring system (PEMS) on two restricted driving tracts. Six hundred eighteen exhaust plumes were collected through the PEMS-chasing measurements from seven trucks. The NO_x emission factors (EFs), and the NO₂/NO_x ratios, were calculated at distinct stages (i.e., tailpipe and on-road). A significant reduction in NO_x EFs (>64%) was observed with normal operating after-treatment devices, except for trucks equipped with diesel particulate filter (DPF). Disparities in tailpipe NO₂/NO_x ratios were also found, attributed to the after-treatment technologies. The NO₂/NO_x ratios measured from plume-chasing were significantly higher (3-4 times, p < 0.001) than the tailpipe measurements, providing field evidence of substantial NO₂ formation in exhaust plumes. We developed a quantitative relationship between NO₂/NO_x ratios from tailpipe and plume-chasing measurements and demonstrated a robust correlation (R² > 0.90). Since the NO₂ formation in the exhaust plume is not explicitly accounted for in NO₂ modeling, the quantitative relationship (O₃-NO₂/NO_x) could improve the estimation of NO₂ exposure when local emission inventory (tailpipe emissions) is available.

Traffic-related air pollutants (TRAPs) emitted from vehicle tailpipes disperse into nearby microenvironments, posing potential exposure risks. Thus, accurately identifying the emission hotspots of TRAPs is essential for assessing potential exposure risks. We investigated the relationship between turbulent kinetic energy (TKE) and pollutant dispersion (D) through an integrated field measurement. A five-year near-road sampling campaign (5 min based) near a light-duty vehicle-restricted roadway and an on-road sampling campaign (5 s based) on isolated proving grounds were conducted. The D was first calculated based on vehicle emission and pollutant concentrations and then paired with TKE measurements. Here, 198 near-road and 377 on-road measurement pairs were collected. In the near-road measurements, TKE and D showed a positive relationship (R² ≥ 0.69) with the vehicle flow rate, while they showed similar decay patterns and sensitivity to vehicle types in the on-road measurements. A relationship between TKE and D (TKE-D) was developed through these measurements, demonstrating a robust correlation (R² ≥ 0.61) and consistent slope values (1.1-1.3). These findings provide field evidence for the positive association between TKE and D, irrespective of the measurement techniques or locations. The TKE-D relationship enables vehicle emission estimation with TKE as the sole input, facilitating the identification of emission hotspots with high spatiotemporal resolution.

Fibromuscular dysplasia (FMD) is a poorly understood disease affecting 3–5% of adult females. The pathobiology of FMD involves arterial lesions of stenosis, dissection, tortuosity, dilation and aneurysm, which can lead to hypertension, stroke, myocardial infarction and even death. Currently, there are no animal models for FMD and few insights as to its pathobiology. In this study, by integrating DNA genotype and RNA sequence data from primary fibroblasts of 83 patients with FMD and 71 matched healthy controls, we inferred 18 gene regulatory co-expression networks, four of which were found to act together as an FMD-associated supernetwork in the arterial wall. After in vivo perturbation of this co-expression supernetwork by selective knockout of a top network key driver, mice developed arterial dilation, a hallmark of FMD. Molecular studies indicated that this supernetwork governs multiple aspects of vascular cell physiology and functionality, including collagen/matrix production. These studies illuminate the complex causal mechanisms of FMD and suggest a potential therapeutic avenue for this challenging disease.

Water-soluble inorganic ions (WSIIs, primarily NH₄⁺, SO₄^2-, and NO₃^-) are major components in ambient PM_2.5, but their reproductive toxicity remains largely unknown. An animal study was conducted where parental mice were exposed to PM_2.5 WSIIs or clean air during preconception and the gestational period. After delivery, all maternal and offspring mice lived in a clean air environment. We assessed reproductive organs, gestation outcome, birth weight, and growth trajectory of the offspring mice. In parallel, we collected birth weight and placenta transcriptome data from 150 mother-infant pairs from the Rhode Island Child Health Study. We found that PM_2.5 WSIIs induced a broad range of adverse reproductive outcomes in mice. PM_2.5 NH₄⁺, SO₄^2-, and NO₃^- exposure reduced ovary weight by 24.22% (p = 0.005), 14.45% (p = 0.048), and 16.64% (p = 0.022) relative to the clean air controls. PM_2.5 SO₄^2- exposure reduced the weight of testicle by 5.24% (p = 0.025); further, mice in the PM_2.5 SO₄^2- exposure group had 1.81 (p = 0.027) fewer offspring than the control group. PM_2.5 NH₄⁺, SO₄^2-, and NO₃^- exposure all led to lower birth than controls. In mice, 557 placenta genes were perturbed by exposure. Integrative analysis of mouse and human data suggested hypoxia response in placenta as an etiological mechanism underlying PM_2.5 WSII exposure's reproductive toxicity.

A well-functioning placenta is essential for fetal and maternal health throughout pregnancy. Using placental weight as a proxy for placental growth, we report genome-wide association analyses in the fetal (n = 65,405), maternal (n = 61,228) and paternal (n = 52,392) genomes, yielding 40 independent association signals. Twenty-six signals are classified as fetal, four maternal and three fetal and maternal. A maternal parent-of-origin effect is seen near KCNQ1. Genetic correlation and colocalization analyses reveal overlap with birth weight genetics, but 12 loci are classified as predominantly or only affecting placental weight, with connections to placental development and morphology, and transport of antibodies and amino acids. Mendelian randomization analyses indicate that fetal genetically mediated higher placental weight is causally associated with preeclampsia risk and shorter gestational duration. Moreover, these analyses support the role of fetal insulin in regulating placental weight, providing a key link between fetal and placental growth.