Common and rare variant associations with clonal haematopoiesis phenotypes.

Clonal haematopoiesis involves the expansion of certain blood cell lineages and has been associated with ageing and adverse health outcomes1-5. Here we use exome sequence data on 628,388 individuals to identify 40,208 carriers of clonal haematopoiesis of indeterminate potential (CHIP). Using genome-wide and exome-wide association analyses, we identify 24 loci (21 of which are novel) where germline genetic variation influences predisposition to CHIP, including missense variants in the lymphocytic antigen coding gene LY75, which are associated with reduced incidence of CHIP. We also identify novel rare variant associations with clonal haematopoiesis and telomere length. Analysis of 5,041 health traits from the UK Biobank (UKB) found relationships between CHIP and severe COVID-19 outcomes, cardiovascular disease, haematologic traits, malignancy, smoking, obesity, infection and all-cause mortality. Longitudinal and Mendelian randomization analyses revealed that CHIP is associated with solid cancers, including non-melanoma skin cancer and lung cancer, and that CHIP linked to DNMT3A is associated with the subsequent development of myeloid but not lymphoid leukaemias. Additionally, contrary to previous findings from the initial 50,000 UKB exomes6, our results in the full sample do not support a role for IL-6 inhibition in reducing the risk of cardiovascular disease among CHIP carriers. Our findings demonstrate that CHIP represents a complex set of heterogeneous phenotypes with shared and unique germline genetic causes and varied clinical implications.

Germline Mutations in CIDEB and Protection against Liver Disease.

BACKGROUND: Exome sequencing in hundreds of thousands of persons may enable the identification of rare protein-coding genetic variants associated with protection from human diseases like liver cirrhosis, providing a strategy for the discovery of new therapeutic targets. METHODS: We performed a multistage exome sequencing and genetic association analysis to identify genes in which rare protein-coding variants were associated with liver phenotypes. We conducted in vitro experiments to further characterize associations. RESULTS: The multistage analysis involved 542,904 persons with available data on liver aminotransferase levels, 24,944 patients with various types of liver disease, and 490,636 controls without liver disease. We found that rare coding variants in APOB, ABCB4, SLC30A10, and TM6SF2 were associated with increased aminotransferase levels and an increased risk of liver disease. We also found that variants in CIDEB, which encodes a structural protein found in hepatic lipid droplets, had a protective effect. The burden of rare predicted loss-of-function variants plus missense variants in CIDEB (combined carrier frequency, 0.7%) was associated with decreased alanine aminotransferase levels (beta per allele, -1.24 U per liter; 95% confidence interval [CI], -1.66 to -0.83; P = 4.8×10-9) and with 33% lower odds of liver disease of any cause (odds ratio per allele, 0.67; 95% CI, 0.57 to 0.79; P = 9.9×10-7). Rare coding variants in CIDEB were associated with a decreased risk of liver disease across different underlying causes and different degrees of severity, including cirrhosis of any cause (odds ratio per allele, 0.50; 95% CI, 0.36 to 0.70). Among 3599 patients who had undergone bariatric surgery, rare coding variants in CIDEB were associated with a decreased nonalcoholic fatty liver disease activity score (beta per allele in score units, -0.98; 95% CI, -1.54 to -0.41 [scores range from 0 to 8, with higher scores indicating more severe disease]). In human hepatoma cell lines challenged with oleate, CIDEB small interfering RNA knockdown prevented the buildup of large lipid droplets. CONCLUSIONS: Rare germline mutations in CIDEB conferred substantial protection from liver disease. (Funded by Regeneron Pharmaceuticals.).

ADAM10 and ADAM17 proteases mediate proinflammatory cytokine-induced and constitutive cleavage of endomucin from the endothelial surface.

Contact between inflammatory cells and endothelial cells (ECs) is a crucial step in vascular inflammation. Recently, we demonstrated that the cell-surface level of endomucin (EMCN), a heavily O-glycosylated single-transmembrane sialomucin, interferes with the interactions between inflammatory cells and ECs. We have also shown that, in response to an inflammatory stimulus, EMCN is cleared from the cell surface by an unknown mechanism. In this study, using adenovirus-mediated overexpression of a tagged EMCN in human umbilical vein ECs, we found that treatment with tumor necrosis factor α (TNF-α) or the strong oxidant pervanadate leads to loss of cell-surface EMCN and increases the levels of the C-terminal fragment of EMCN 3- to 4-fold. Furthermore, treatment with the broad-spectrum matrix metalloproteinase inhibitor batimastat (BB94) or inhibition of ADAM metallopeptidase domain 10 (ADAM10) and ADAM17 with two small-molecule inhibitors, GW280264X and GI254023X, or with siRNA significantly reduced basal and TNFα-induced cell-surface EMCN cleavage. Release of the C-terminal fragment of EMCN by TNF-α treatment was blocked by chemical inhibition of ADAM10 alone or in combination with ADAM17. These results indicate that cell-surface EMCN undergoes constitutive cleavage and that TNF-α treatment dramatically increases this cleavage, which is mediated predominantly by ADAM10 and ADAM17. As endothelial cell-surface EMCN attenuates leukocyte-EC interactions during inflammation, we propose that EMCN is a potential therapeutic target to manage vascular inflammation.

Cell-specific expression of lung disease risk-related genes in the human small airway epithelium.

BACKGROUND: The human small airway epithelium (SAE) plays a central role in the early events in the pathogenesis of most inherited and acquired lung disorders. Little is known about the molecular phenotypes of the specific cell populations comprising the SAE in humans, and the contribution of SAE specific cell populations to the risk for lung diseases. METHODS: Drop-seq single-cell RNA-sequencing was used to characterize the transcriptome of single cells from human SAE of nonsmokers and smokers by bronchoscopic brushing. RESULTS: Eleven distinct cell populations were identified, including major and rare epithelial cells, and immune/inflammatory cells. There was cell type-specific expression of genes relevant to the risk of the inherited pulmonary disorders, genes associated with risk of chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis and (non-mutated) driver genes for lung cancers. Cigarette smoking significantly altered the cell type-specific transcriptomes and disease risk-related genes. CONCLUSIONS: This data provides new insights into the possible contribution of specific lung cells to the pathogenesis of lung disorders.

Glycocalyx regulation of vascular endothelial growth factor receptor 2 activity.

We have previously shown that knockdown of endomucin (EMCN), an integral membrane glycocalyx glycoprotein, prevents VEGF-induced proliferation, migration, and tube formation in vitro and angiogenesis in vivo. In the endothelium, VEGF mediates most of its angiogenic effects through VEGF receptor 2 (VEGFR2). To understand the role of EMCN, we examined the effect of EMCN depletion on VEGFR2 endocytosis and activation. Results showed that although VEGF stimulation promoted VEGFR2 internalization in control endothelial cells (ECs), loss of EMCN prevented VEGFR2 endocytosis. Cell surface analysis revealed a decrease in VEGFR2 following VEGF stimulation in control but not siRNA directed against EMCN-transfected ECs. EMCN depletion resulted in heightened phosphorylation following VEGF stimulation with an increase in total VEGFR2 protein. These results indicate that EMCN modulates VEGFR2 endocytosis and activity and point to EMCN as a potential therapeutic target.-LeBlanc, M. E., Saez-Torres, K. L., Cano, I., Hu, Z., Saint-Geniez, M., Ng, Y.-S., D'Amore, P. A. Glycocalyx regulation of vascular endothelial growth factor receptor 2 activity.

Anti-secretogranin III therapy of oxygen-induced retinopathy with optimal safety.

Retinopathy of prematurity (ROP) with pathological retinal neovascularization is the most common cause of blindness in children. ROP is currently treated with laser therapy or cryotherapy, both of which may adversely affect the peripheral vision with limited efficacy. Owing to the susceptibility of the developing retina and vasculatures to pharmacological intervention, there is currently no approved drug therapy for ROP in preterm infants. Secretogranin III (Scg3) was recently discovered as a highly disease-restricted angiogenic factor, and a Scg3-neutralizing monoclonal antibody (mAb) was reported with high efficacy to alleviate oxygen-induced retinopathy (OIR) in mice, a surrogate model of ROP. Herein we independently investigated the efficacy of anti-Scg3 mAb in OIR mice and characterized its safety in neonatal mice. We developed a new Scg3-neutralizing mAb recognizing a distinct epitope and independently established the therapeutic activity of anti-Scg3 therapy to alleviate OIR-induced pathological retinal neovascularization in mice. Importantly, anti-Scg3 mAb showed no detectable adverse effects on electroretinography and developing retinal vasculature. Furthermore, systemic anti-Scg3 mAb induced no renal tubular injury or abnormality in kidney vessel development and body weight gain of neonatal mice. In contrast, anti-vascular endothelial growth factor drug aflibercept showed significant side effects in neonatal mice. These results suggest that anti-Scg3 mAb may have the safety and efficacy profiles required for ROP therapy.

Secretogranin III as a novel target for the therapy of choroidal neovascularization.

Wet age-related macular degeneration (AMD) with choroidal neovascularization (CNV) is a leading cause of vision loss in the elderly. The advent of anti-vascular endothelial growth factor (VEGF) drugs represents a major breakthrough in wet AMD therapy but with limited efficacy to improve visual acuity. Secretogranin III (Scg3, SgIII) was recently discovered as a novel angiogenic factor with VEGF-independent mechanisms. Scg3-neutralizing monoclonal antibody (mAb) was reported to alleviate pathological retinal neovascularization in oxygen-induced retinopathy mice and retinal vascular leakage in diabetic mice with high efficacy and disease selectivity. Herein we investigated whether Scg3 is a novel angiogenic target for CNV therapy in mouse models. We found that anti-Scg3 ML49.3 mAb inhibited Scg3-induced proliferation and Src phosphorylation in human retinal microvascular endothelial cells. Intravitreal injection of Scg3-neutralizing polyclonal antibodies (pAb) or mAb significantly attenuated laser-induced CNV leakage, CNV 3D volume, lesion area and vessel density. Furthermore, subcutaneous administration of Scg3-neutralizing pAb or mAb significantly prevented Matrigel-induced CNV. The efficacy of anti-Scg3 pAb or mAb was comparable to VEGF inhibitor aflibercept. These findings suggest that Scg3 plays an important role in CNV pathogenesis and that anti-Scg3 mAb efficiently ameliorates laser- or Matrigel-induced CNV.

Secretogranin III: a diabetic retinopathy-selective angiogenic factor.

Secretogranin III (Scg3) is a member of the granin protein family that regulates the biogenesis of secretory granules. Scg3 was recently discovered as an angiogenic factor, expanding its functional role to extrinsic regulation. Unlike many other known angiogenic factors, the pro-angiogenic actions of Scg3 are restricted to pathological conditions. Among thousands of quantified endothelial ligands, Scg3 has the highest binding activity ratio to diabetic vs. healthy mouse retinas and lowest background binding to normal vessels. In contrast, vascular endothelial growth factor binds to and stimulates angiogenesis of both diabetic and control vasculature. Consistent with its role in pathological angiogenesis, Scg3-neutralizing antibodies alleviate retinal vascular leakage in mouse models of diabetic retinopathy and retinal neovascularization in oxygen-induced retinopathy mice. This review summarizes our current knowledge of Scg3 as a regulatory protein of secretory granules, highlights its new role as a highly disease-selective angiogenic factor, and envisions Scg3 inhibitors as "selective angiogenesis blockers" for targeted therapy.

Pathogenic role and therapeutic potential of pleiotrophin in mouse models of ocular vascular disease.

Angiogenic factors play an important role in the pathogenesis of diabetic retinopathy (DR), neovascular age-related macular degeneration (nAMD) and retinopathy of prematurity (ROP). Pleiotrophin, a well-known angiogenic factor, was recently reported to be upregulated in the vitreous fluid of patients with proliferative DR (PDR). However, its pathogenic role and therapeutic potential in ocular vascular diseases have not been defined in vivo. Here using corneal pocket assays, we demonstrated that pleiotrophin induced angiogenesis in vivo. To investigate the pathological role of pleiotrophin we used neutralizing antibody to block its function in multiple in vivo models of ocular vascular diseases. In a mouse model of DR, intravitreal injection of pleiotrophin-neutralizing antibody alleviated diabetic retinal vascular leakage. In a mouse model of oxygen-induced retinopathy (OIR), which is a surrogate model of ROP and PDR, we demonstrated that intravitreal injection of anti-pleiotrophin antibody prevented OIR-induced pathological retinal neovascularization and aberrant vessel tufts. Finally, pleiotrophin-neutralizing antibody ameliorated laser-induced choroidal neovascularization, a mouse model of nAMD, suggesting that pleiotrophin is involved in choroidal vascular disease. These findings suggest that pleiotrophin plays an important role in the pathogenesis of DR with retinal vascular leakage, ROP with retinal neovascularization and nAMD with choroidal neovascularization. The results also support pleiotrophin as a promising target for anti-angiogenic therapy.

The regulatory role of hepatoma-derived growth factor as an angiogenic factor in the eye.

PURPOSE: Hepatoma-derived growth factor (HDGF) is a mitogen that promotes endothelial proliferation and neuronal survival. Using a unique technology of ligandomics, we recently identified HDGF as a retinal endothelial binding protein. The purpose of this study is to examine the role of HDGF in regulating ocular vasculature and the expression of HDGF in the retina. METHODS: HDGF expression in the retinal was analyzed with western blot and immunohistochemistry. Angiogenic activity was investigated in human retinal microvascular endothelial cells (HRMVECs) with in vitro endothelial proliferation, migration, and permeability assays. In vivo angiogenic activity was quantified with a corneal pocket assay. The Evans blue assay and western blot using anti-mouse albumin were performed to detect the capacity of HDGF to induce retinal vascular leakage. RESULTS: Immunohistochemistry revealed that HDGF is expressed in the retina with a distinct pattern. HDGF was detected in retinal ganglion cells and the inner nuclear layer but not in the inner plexiform layer, suggesting that HDGF is expressed in the nucleus, but not in the cytoplasm, of retinal neurons. In contrast to family member HDGF-related protein 3 (HRP-3) that has no expression in photoreceptors, HDGF is also present in the outer nuclear layer and the inner and outer segments of photoreceptors. This suggests that HDGF is expressed in the nucleus as well as the cytoplasm of photoreceptors. In vitro functional assays showed that HDGF induced the proliferation, migration, and permeability of HRMVECs. Corneal pocket assay indicated that HDGF directly stimulated angiogenesis in vivo. Intravitreal injection of HDGF significantly induced retinal vascular leakage. CONCLUSIONS: These results suggest that HDGF is an angiogenic factor that regulates retinal vasculature in physiologic and pathological conditions. Identification of HDGF by ligandomics and its independent characterization in this study also support the validity of this new technology for systematic identification of cellular ligands, including angiogenic factors.

Mesd extrinsically promotes phagocytosis by retinal pigment epithelial cells.

Phagocytosis is a critical process to maintain tissue homeostasis. In the retina, photoreceptor cells renew their photoexcitability by shedding photoreceptor outer segments (POSs) in a diurnal rhythm. Shed POSs are phagocytosed by retinal pigment epithelial (RPE) cells to prevent debris accumulation, retinal degeneration, and blindness. Phagocytosis ligands are the key to understanding how RPE recognizes shed POSs. Here, we characterized mesoderm development candidate 2 (Mesd or Mesdc2), an endoplasmic reticulum (ER) chaperon for low-density lipoprotein receptor-related proteins (LRPs), to extrinsically promote RPE phagocytosis. The results showed that Mesd stimulated phagocytosis of fluorescence-labeled POS vesicles by D407 RPE cells. Ingested POSs were partially degraded within 3 h in some RPE cells to dispense undegradable fluorophore throughout the cytoplasm. Internalized POSs were colocalized with phagosome biomarker Rab7, suggesting that Mesd-mediated engulfment is involved in a phagocytosis pathway. Mesd also facilitated phagocytosis of POSs by primary RPE cells. Mesd bound to unknown phagocytic receptor(s) on RPE cells. Mesd was detected in the cytoplasm, but not nuclei, of different retinal layers and is predominantly expressed in the ER-free cellular compartment of POSs. Mesd was not secreted into medium from healthy cells but passively released from apoptotic cells with increased membrane permeability. Released Mesd selectively bound to the surface of POS vesicles and apoptotic cells, but not healthy cells. These results suggest that Mesd may be released from and bind to shed POSs to facilitate their phagocytic clearance.

Lyar Is a New Ligand for Retinal Pigment Epithelial Phagocytosis.

Phagocytosis is critical to tissue homeostasis, as highlighted by phagocytosis defect of retinal pigment epithelial (RPE) cells with debris accumulation, photoreceptor degeneration and blindness. Phagocytosis ligands are the key to delineating molecular mechanisms and functional roles of phagocytes, but are traditionally identified in individual cases with technical challenges. We recently developed open reading frame phage display (OPD) for phagocytosis-based functional cloning (PFC) to identify unknown ligands. One of the identified ligands was Ly-1 antibody reactive clone (Lyar) with functions poorly defined. Herein, we characterized Lyar as a new ligand to stimulate RPE phagocytosis. In contrast to its reported nucleolar expression, immunohistochemistry showed that Lyar was highly expressed in photoreceptor outer segments (POSs) of the retina. Cytoplasmic Lyar was released from apoptotic cells, and selectively bound to shed POSs and apoptotic cells, but not healthy cells. POS vesicles engulfed through Lyar-dependent pathway were targeted to phagosomes and colocalized with phagosome marker Rab7. These results suggest that Lyar is a genuine RPE phagocytosis ligand, which in turn supports the validity of OPD/PFC as the only available approach for unbiased identification of phagocytosis ligands with broad applicability to various phagocytes.

Interventions to support children's engagement in health-related decisions: a systematic review.

BACKGROUND: Children often need support in health decision-making. The objective of this study was to review characteristics and effectiveness of interventions that support health decision-making of children. METHODS: A systematic review. Electronic databases (PubMed, the Cochrane Library, Web of Science, Scopus, ProQuest Dissertations and Theses, CINAHL, PsycINFO, MEDLINE, and EMBASE) were searched from inception until March 2012. Two independent reviewers screened eligibility: a) intervention studies; b) involved supporting children (≤18 years) considering health-related decision(s); and c) measured decision quality or decision-making process outcomes. Data extraction and quality appraisal were conducted by one author and verified by another using a standardized data extraction form. Quality appraisal was based on the Cochrane Risk of Bias tool. RESULTS: Of 4313 citations, 5 studies were eligible. Interventions focused on supporting decisions about risk behaviors (n = 3), psycho-educational services (n = 1), and end of life (n = 1). Two of 5 studies had statistically significant findings: i) compared to attention placebo, decision coaching alone increased values congruence between child and parent, and child satisfaction with decision-making process (lower risk of bias); ii) compared to no intervention, a workshop with weekly assignments increased overall decision-making quality (higher risk of bias). CONCLUSIONS: Few studies have focused on interventions to support children's participation in decisions about their health. More research is needed to determine effective methods for supporting children's health decision-making.

Prioritizing Solutions and Improving Resources among Young Pediatric Brain Tumor Survivors: Results of an Online Survey.

Pediatric Brain Tumor Survivors (PBTS) often experience social, academic and employment difficulties during aftercare. Despite their needs, they often do not use the services available to them. Following a previous qualitative study, we formulated solutions to help support PBTS return to daily activities after treatment completion. The present study aims to confirm and prioritize these solutions with a larger sample. We used a mixed-methods survey with 68 participants (43 survivors, 25 parents, PBTS' age: 15-39 years). Firstly, we collected information about health condition, and school/work experience in aftercare. Then, we asked participants to prioritize the previously identified solutions using Likert scales and open-ended questions. We used descriptive and inferential statistics to analyze data, and qualitative information to support participants' responses. Participants prioritized the need for evaluation, counseling, and follow-up by health professionals to better understand their post-treatment needs, obtain help to access adapted services, and receive information about resources at school/work. Responses to open-ended questions highlighted major challenges regarding the implementation of professionals' recommendations at school/work and the need for timely interventions. These results will help refine solutions for PBTS and provide key elements for future implementation. Translating these priorities into action will need further work involving professionals and decision makers.

A large meta-analysis identifies genes associated with anterior uveitis.

Anterior Uveitis (AU) is the inflammation of the anterior part of the eye, the iris and ciliary body and is strongly associated with HLA-B*27. We report AU exome sequencing results from eight independent cohorts consisting of 3,850 cases and 916,549 controls. We identify common genome-wide significant loci in HLA-B (OR = 3.37, p = 1.03e-196) and ERAP1 (OR = 0.86, p = 1.1e-08), and find IPMK (OR = 9.4, p = 4.42e-09) and IDO2 (OR = 3.61, p = 6.16e-08) as genome-wide significant genes based on the burden of rare coding variants. Dividing the cohort into HLA-B*27 positive and negative individuals, we find ERAP1 haplotype is strongly protective only for B*27-positive AU (OR = 0.73, p = 5.2e-10). Investigation of B*27-negative AU identifies a common signal near HLA-DPB1 (rs3117230, OR = 1.26, p = 2.7e-08), risk genes IPMK and IDO2, and several additional candidate risk genes, including ADGFR5, STXBP2, and ACHE. Taken together, we decipher the genetics underlying B*27-positive and -negative AU and identify rare and common genetic signals for both subtypes of disease.

Converging evidence from exome sequencing and common variants implicates target genes for osteoporosis.

In this study, we leveraged the combined evidence of rare coding variants and common alleles to identify therapeutic targets for osteoporosis. We undertook a large-scale multiancestry exome-wide association study for estimated bone mineral density, which showed that the burden of rare coding alleles in 19 genes was associated with estimated bone mineral density (P < 3.6 × 10-7). These genes were highly enriched for a set of known causal genes for osteoporosis (65-fold; P = 2.5 × 10-5). Exome-wide significant genes had 96-fold increased odds of being the top ranked effector gene at a given GWAS locus (P = 1.8 × 10-10). By integrating proteomics Mendelian randomization evidence, we prioritized CD109 (cluster of differentiation 109) as a gene for which heterozygous loss of function is associated with higher bone density. CRISPR-Cas9 editing of CD109 in SaOS-2 osteoblast-like cell lines showed that partial CD109 knockdown led to increased mineralization. This study demonstrates that the convergence of common and rare variants, proteomics and CRISPR can highlight new bone biology to guide therapeutic development.

Rare coding variants in CHRNB2 reduce the likelihood of smoking.

Human genetic studies of smoking behavior have been thus far largely limited to common variants. Studying rare coding variants has the potential to identify drug targets. We performed an exome-wide association study of smoking phenotypes in up to 749,459 individuals and discovered a protective association in CHRNB2, encoding the ß2 subunit of the α4ß2 nicotine acetylcholine receptor. Rare predicted loss-of-function and likely deleterious missense variants in CHRNB2 in aggregate were associated with a 35% decreased odds for smoking heavily (odds ratio (OR) = 0.65, confidence interval (CI) = 0.56-0.76, P = 1.9 × 10-8). An independent common variant association in the protective direction ( rs2072659 ; OR = 0.96; CI = 0.94-0.98; P = 5.3 × 10-6) was also evident, suggesting an allelic series. Our findings in humans align with decades-old experimental observations in mice that ß2 loss abolishes nicotine-mediated neuronal responses and attenuates nicotine self-administration. Our genetic discovery will inspire future drug designs targeting CHRNB2 in the brain for the treatment of nicotine addiction.

Multiancestry exome sequencing reveals INHBE mutations associated with favorable fat distribution and protection from diabetes.

Body fat distribution is a major, heritable risk factor for cardiometabolic disease, independent of overall adiposity. Using exome-sequencing in 618,375 individuals (including 160,058 non-Europeans) from the UK, Sweden and Mexico, we identify 16 genes associated with fat distribution at exome-wide significance. We show 6-fold larger effect for fat-distribution associated rare coding variants compared with fine-mapped common alleles, enrichment for genes expressed in adipose tissue and causal genes for partial lipodystrophies, and evidence of sex-dimorphism. We describe an association with favorable fat distribution (p = 1.8 × 10-09), favorable metabolic profile and protection from type 2 diabetes (~28% lower odds; p = 0.004) for heterozygous protein-truncating mutations in INHBE, which encodes a circulating growth factor of the activin family, highly and specifically expressed in hepatocytes. Our results suggest that inhibin ßE is a liver-expressed negative regulator of adipose storage whose blockade may be beneficial in fat distribution-associated metabolic disease.

Smoking shifts human small airway epithelium club cells toward a lesser differentiated population.

The club cell, a small airway epithelial (SAE) cell, plays a central role in human lung host defense. We hypothesized that subpopulations of club cells with distinct functions may exist. The SAE of healthy nonsmokers and healthy cigarette smokers were evaluated by single-cell RNA sequencing, and unsupervised clustering revealed subpopulations of SCGCB1A1+KRT5loMUC5AC- club cells. Club cell heterogeneity was supported by evaluations of SAE tissue sections, brushed SAE cells, and in vitro air-liquid interface cultures. Three subpopulations included: (1) progenitor; (2) proliferating; and (3) effector club cells. The progenitor club cell population expressed high levels of mitochondrial, ribosomal proteins, and KRT5 relative to other club cell populations and included a differentiation branch point leading to mucous cell production. The small proliferating population expressed high levels of cyclins and proliferation markers. The effector club cell cluster expressed genes related to host defense, xenobiotic metabolism, and barrier functions associated with club cell function. Comparison of smokers vs. nonsmokers demonstrated that smoking limited the extent of differentiation of all three subclusters and altered SAM pointed domain-containing Ets transcription factor (SPDEF)-regulated transcription in the effector cell population leading to a change in the location of the branch point for mucous cell production, a potential explanation for the concomitant reduction in effector club cells and increase in mucous cells in smokers. These observations provide insights into both the makeup of human SAE club cell subpopulations and the smoking-induced changes in club cell biology.