Joint genotype and ancestry analysis identify novel loci associated with atopic dermatitis in African American population.

Atopic dermatitis (AD) is a chronic itchy inflammatory disease of the skin. Genetic studies have identified multiple risk factors linked to the disease; however, most of the studies have been derived from European and East Asian populations. The admixed African American (AA) genome may provide an opportunity to discovery ancestry-specific loci involved in AD susceptibility. Herein, we present joint analysis of ancestry and genotype effects followed by validation using differential gene expression analysis on AD using 726 AD-affected individuals and 999 non-AD control individuals from the AA population, genotyped using Multi-Ethnic Global Array (MEGA) followed by imputation using the Consortium on Asthma among African Ancestry Populations in the Americas (CAAPA) reference panel. The joint analysis identified two novel AD-susceptibility loci, rs2195989 in gene ANGPT1 (8q23.1) and rs62538818 in the intergenic region between genes LURAP1L and MPDZ (9p23). Admixture mapping (AM) results showed potential genomic inflation, and we implemented genomic control and identified five ancestry-of-origin loci with European ancestry effects. The multi-omics functional prioritization of variants in AM signals prioritized the loci SLAIN2, RNF39, and FOXA2. Genome-wide association study (GWAS) identified variants significantly associated with AD in the AA population, including SGK1 (rs113357522, odds ratio [OR] = 2.81), EFR3A (rs16904552, OR = 1.725), and MMP14 (rs911912, OR = 1.791). GWAS variants were common in the AA but rare in the European population, which suggests an African-ancestry-specific risk of AD. Four genes (ANGPT1, LURAP1L, EFR3A, and SGK1) were further validated using qPCR from AD and healthy skin. This study highlighted the importance of genetic studies on admixed populations, as well as local ancestry and genotype-ancestry joint effects to identify risk loci for AD.

Regulation of MYC by CARD14 in human epithelium is a determinant of epidermal homeostasis and disease.

Caspase recruitment domain family member 14 (CARD14) and its variants are associated with both atopic dermatitis (AD) and psoriasis, but their mechanistic impact on skin barrier homeostasis is largely unknown. CARD14 is known to signal via NF-κB; however, CARD14-NF-κB signaling does not fully explain the heterogeneity of CARD14-driven disease. Here, we describe a direct interaction between CARD14 and MYC and show that CARD14 signals through MYC in keratinocytes to coordinate skin barrier homeostasis. CARD14 directly binds MYC and influences barrier formation in an MYC-dependent fashion, and this mechanism is undermined by disease-associated CARD14 variants. These studies establish a paradigm that CARD14 activation regulates skin barrier function by two distinct mechanisms, including activating NF-κB to bolster the antimicrobial (chemical) barrier and stimulating MYC to bolster the physical barrier. Finally, we show that CARD14-dependent MYC signaling occurs in other epithelia, expanding the impact of our findings beyond the skin.

Filaggrin loss-of-function variants are associated with atopic dermatitis phenotypes in a diverse, early-life prospective cohort.

Loss-of-function (LoF) variants in the filaggrin (FLG) gene are the strongest known genetic risk factor for atopic dermatitis (AD), but the impact of these variants on AD outcomes is poorly understood. We comprehensively identified genetic variants through targeted region sequencing of FLG in children participating in the Mechanisms of Progression of Atopic Dermatitis to Asthma in Children cohort. Twenty FLG LoF variants were identified, including 1 novel variant and 9 variants not previously associated with AD. FLG LoF variants were found in the cohort. Among these children, the presence of 1 or more FLG LoF variants was associated with moderate/severe AD compared with those with mild AD. Children with FLG LoF variants had a higher SCORing for Atopic Dermatitis (SCORAD) and higher likelihood of food allergy within the first 2.5 years of life. LoF variants were associated with higher transepidermal water loss (TEWL) in both lesional and nonlesional skin. Collectively, our study identifies established and potentially novel AD-associated FLG LoF variants and associates FLG LoF variants with higher TEWL in lesional and nonlesional skin.

Birth outcomes in relation to neighborhood food access and individual food insecurity during pregnancy in the Environmental Influences on Child Health Outcomes (ECHO)-wide cohort study.

BACKGROUND: Limited access to healthy foods, resulting from residence in neighborhoods with low-food access or from household food insecurity, is a public health concern. Contributions of these measures during pregnancy to birth outcomes remain understudied. OBJECTIVES: We examined associations between neighborhood food access and individual food insecurity during pregnancy with birth outcomes. METHODS: We used data from 53 cohorts participating in the nationwide Environmental Influences on Child Health Outcomes-Wide Cohort Study. Participant inclusion required a geocoded residential address or response to a food insecurity question during pregnancy and information on birth outcomes. Exposures include low-income-low-food-access (LILA, where the nearest supermarket is >0.5 miles for urban or >10 miles for rural areas) or low-income-low-vehicle-access (LILV, where few households have a vehicle and >0.5 miles from the nearest supermarket) neighborhoods and individual food insecurity. Mixed-effects models estimated associations with birth outcomes, adjusting for socioeconomic and pregnancy characteristics. RESULTS: Among 22,206 pregnant participants (mean age 30.4 y) with neighborhood food access data, 24.1% resided in LILA neighborhoods and 13.6% in LILV neighborhoods. Of 1630 pregnant participants with individual-level food insecurity data (mean age 29.7 y), 8.0% experienced food insecurity. Residence in LILA (compared with non-LILA) neighborhoods was associated with lower birth weight [ß -44.3 g; 95% confidence interval (CI): -62.9, -25.6], lower birth weight-for-gestational-age z-score (-0.09 SD units; -0.12, -0.05), higher odds of small-for-gestational-age [odds ratio (OR) 1.15; 95% CI: 1.00, 1.33], and lower odds of large-for-gestational-age (0.85; 95% CI: 0.77, 0.94). Similar findings were observed for residence in LILV neighborhoods. No associations of individual food insecurity with birth outcomes were observed. CONCLUSIONS: Residence in LILA or LILV neighborhoods during pregnancy is associated with adverse birth outcomes. These findings highlight the need for future studies examining whether investing in neighborhood resources to improve food access during pregnancy would promote equitable birth outcomes.

Progressive accumulation of hyperinflammatory NKG2Dlow NK cells in early childhood severe atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that often precedes the development of food allergy, asthma, and allergic rhinitis. The prevailing paradigm holds that a reduced frequency and function of natural killer (NK) cell contributes to AD pathogenesis, yet the underlying mechanisms and contributions of NK cells to allergic comorbidities remain ill-defined. Here, analysis of circulating NK cells in a longitudinal early life cohort of children with AD revealed a progressive accumulation of NK cells with low expression of the activating receptor NKG2D, which was linked to more severe AD and sensitivity to allergens. This was most notable in children co-sensitized to food and aeroallergens, a risk factor for development of asthma. Individual-level longitudinal analysis in a subset of children revealed coincident reduction of NKG2D on NK cells with acquired or persistent sensitization, and this was associated with impaired skin barrier function assessed by transepidermal water loss. Low expression of NKG2D on NK cells was paradoxically associated with depressed cytolytic function but exaggerated release of the proinflammatory cytokine tumor necrosis factor-α. These observations provide important insights into a potential mechanism underlying the development of allergic comorbidity in early life in children with AD, which involves altered NK cell functional responses, and define an endotype of severe AD.

Temporal relationships between Staphylococcus aureus colonization, filaggrin expression, and pediatric atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is characterized by Staphylococcus aureus (S. aureus) colonization. Longitudinal early life data delineating relationships of S. aureus colonization, barrier function, and AD outcomes are lacking. We define longitudinal S. aureus endotypes and AD pathogenesis in early life. METHODS: We defined longitudinal S. aureus skin colonization phenotypes across two annual visits (non-colonized: V1- V2- , early transient: V1+ V2- , late-onset: V1- V2+ , persistent: V1+ V2+ ) in the Mechanisms of Progression of Atopic Dermatitis to Asthma in Children cohort. We analyzed AD severity, sensitization, and skin barrier function across phenotypes, and performed mediation analyses between colonization and FLG expression. RESULTS: Persistent S. aureus colonization was associated with increased SCORAD at V1 (33.5 vs. 19.0, p = .004) and V2 (40.1 vs.16.9, p < .001), and lower non-lesional (NL) FLG at V2 (1.77 vs. 4.09, p = .029) compared to the non-colonized phenotype, with early transient and late-onset colonization as intermediate phenotypes. Children colonized at V2 demonstrated a decrease in NL-FLG expression from V1 to V2 compared to those non-colonized at V2 (p = .0012), who maintained expression. This effect remained significant even after adjusting for V1 colonization and SCORAD (p = .011). CONCLUSIONS: Our findings are the first to present longitudinal quantitative FLG expression and S. aureus skin colonization in early life and suggest that a decrease in NL-FLG drives later colonization. Hence, therapies to maintain NL-FLG expression may prevent S. aureus colonization. Further, a longitudinal AD endotype of persistent colonization is characterized by increased AD severity, sensitization, and decreasing NL-FLG.

Progressive accumulation of hyperinflammatory NKG2D low NK cells in early childhood severe atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that often precedes the development of food allergy, asthma, and allergic rhinitis. The prevailing paradigm holds that a reduced frequency and function of natural killer (NK) cell contributes to AD pathogenesis, yet the underlying mechanisms and contributions of NK cells to allergic co-morbidities remain ill-defined. Herein, analysis of circulating NK cells in a longitudinal early life cohort of children with AD revealed a progressive accumulation of NK cells with low expression of the activating receptor NKG2D, which was linked to more severe AD and sensitivity to allergens. This was most notable in children co-sensitized to food and aero allergens, a risk factor for development of asthma. Individual-level longitudinal analysis in a subset of children revealed co-incident reduction of NKG2D on NK cells with acquired or persistent sensitization, and this was associated with impaired skin barrier function assessed by transepidermal water loss. Low expression of NKG2D on NK cells was paradoxically associated with depressed cytolytic function but exaggerated release of the proinflammatory cytokine TNF-α. These observations provide important insights into a potential mechanism underlying the development of allergic co-morbidity in early life in children with AD which involves altered NK-cell functional responses, and define an endotype of severe AD.

Airway transcriptome networks identify susceptibility to frequent asthma exacerbations in children.

BACKGROUND: Frequent asthma exacerbators, defined as those experiencing more than 1 hospitalization in a year for an asthma exacerbation, represent an important subgroup of individuals with asthma. However, this group remains poorly defined and understudied in children. OBJECTIVE: Our aim was to determine the molecular mechanisms underlying asthma pathogenesis and exacerbation frequency. METHODS: We performed RNA sequencing of upper airway cells from both frequent and nonfrequent exacerbators enrolled in the Ohio Pediatric Asthma Repository. RESULTS: Through molecular network analysis, we found that nonfrequent exacerbators display an increase in modules enriched for immune system processes, including type 2 inflammation and response to infection. In contrast, frequent exacerbators showed expression of modules enriched for nervous system processes, such as synaptic formation and axonal outgrowth. CONCLUSION: These data suggest that the upper airway of frequent exacerbators undergoes peripheral nervous system remodeling, representing a novel mechanism underlying pediatric asthma exacerbation.

B cell repertoire in children with skin barrier dysfunction supports altered IgE maturation associated with allergic food sensitization.

The skin is a major immune organ and skin barrier dysfunction is a major risk factor for the development of the inappropriate immune response seen in allergic disease. Skin barrier disruption alters the landscape of antigens experienced by the immune system and the downstream impacts on the antibody repertoire remain poorly characterized, particularly for the IgE isotype responsible for allergic specificity and in early life, when allergic disease is developing. In this study, we sequenced antibody gene repertoires from a large and well-characterized cohort of children with atopic dermatitis and found that food sensitization was associated with lower mutation frequencies in the IgE compartment. This trend was abrogated in children living with pets during the first year of life. These results elucidate potential molecular mechanisms underlying the protective effects of pet ownership and non-antiseptic environs reported for allergic disease, and the hygiene hypothesis more broadly. We also observed increased IgE diversity and increased isotype-switching to the IgE isotype, suggesting that B cell development, particularly isotype-switching, is heavily altered in the those with food allergen sensitizations relative to those without food allergen sensitizations. Unlike for food antigens, aeroallergen sensitization exhibited no effect on IgE mutation or diversity. Consistent patterns of antibody rearrangement were associated with food allergen sensitization in subjects with atopic dermatitis. Thus, we propose the Immune Repertoire in Atopic Disease (IRAD) score, to quantify this repertoire shift and to aid clinically in patient diagnosis and risk stratification.

Racial Fairness in Precision Medicine: Pediatric Asthma Prediction Algorithms.

PURPOSE: Quantify and examine the racial fairness of two widely used childhood asthma predictive precision medicine algorithms: the asthma predictive index (API) and the pediatric asthma risk score (PARS). DESIGN: Apply the API and PARS and evaluate model performance overall and when stratified by race. SETTING: Cincinnati, OH, USA. SUBJECTS: A prospective birth cohort of 590 children with clinically measured asthma diagnosis by age seven. MEASURES: Model diagnostic criteria included sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). ANALYSIS: Significant differences in model performance between Black and white children were considered to be present if the P-value associated with a t-test based on 100 bootstrap replications was less than .05. RESULTS: Compared to predictions for white children, predictions for Black children using the PARS had a higher sensitivity (.88 vs .57), lower specificity (.55 vs .83), higher PPV (.42 vs .33), but a similar NPV (.93 vs .93). Within the API and compared to predictions for white children, predictions for Black children had a higher sensitivity (.63 vs .53), similar specificity (.81 vs .80), higher PPV (.54 vs .28), and lower NPV (.86 vs .92). CONCLUSIONS: Overall, racial disparities in model diagnostic criteria were greatest for sensitivity and specificity in the PARS, but racial disparities existed in three of the four criteria for both the PARS and the API.

Performance of the Pediatric Asthma Risk Score across Diverse Populations.

BACKGROUND: Methods to determine whether a toddler is likely to develop asthma are of value to parents and clinical trialists testing primary prevention strategies. The Pediatric Asthma Risk Score (PARS) is a 14-point score of six factors designed to predict asthma in early life. PARS was developed and validated in relatively homogenous populations, so its generalizability is unknown. METHODS: We computed PARS using the six factors of self-declared race (parent-reported as "Black" or "not Black"), parental asthma, eczema, any wheezing, wheezing without a cold, and polysensitization in 5634 children from birth to 3 years of age. The primary outcome of our analysis was the ability of PARS to predict asthma development at 5 to 10 years of age using the area under the receiver operating curve in each cohort and across all cohorts with varying ethnicity, sex, cohort type, birth decades, missing PARS factors, and polysensitization definition. We also performed a meta-analysis across all the cohorts. Finally, we compared PARS predictive ability with the binary Asthma Predictive Index (API). RESULTS: Across 10 cohorts, the area under the receiver operating curve for PARS was 0.76. PARS performance did not differ by ethnicity, sex, cohort type, enrollment decade, missing PARS factors, or polysensitization definition (all P>0.05). The weights of each factor in the meta-analysis were similar to the original PARS weights. PARS and API equally identified children at high risk for developing asthma or not; API missed 31% of children at moderate asthma risk. CONCLUSIONS: PARS provided robust estimates of asthma risk in children from a wide range of ethnicities, backgrounds, and susceptibility. (Funded by the National Institute of Allergy and Infectious Diseases and others.)

Multiancestral polygenic risk score for pediatric asthma.

BACKGROUND: Asthma is the most common chronic condition in children and the third leading cause of hospitalization in pediatrics. The genome-wide association study catalog reports 140 studies with genome-wide significance. A polygenic risk score (PRS) with predictive value across ancestries has not been evaluated for this important trait. OBJECTIVES: This study aimed to train and validate a PRS relying on genetic determinants for asthma to provide predictions for disease occurrence in pediatric cohorts of diverse ancestries. METHODS: This study applied a Bayesian regression framework method using the Trans-National Asthma Genetic Consortium genome-wide association study summary statistics to derive a multiancestral PRS score, used one Electronic Medical Records and Genomics (eMERGE) cohort as a training set, used a second independent eMERGE cohort to validate the score, and used the UK Biobank data to replicate the findings. A phenome-wide association study was performed using the PRS to identify shared genetic etiology with other phenotypes. RESULTS: The multiancestral asthma PRS was associated with asthma in the 2 pediatric validation datasets. Overall, the multiancestral asthma PRS has an area under the curve (AUC) of 0.70 (95% CI, 0.69-0.72) in the pediatric validation 1 and AUC of 0.66 (0.65-0.66) in the pediatric validation 2 datasets. We found significant discrimination across pediatric subcohorts of European (AUC, 95% CI, 0.60 and 0.66), African (AUC, 95% CI, 0.61 and 0.66), admixed American (AUC, 0.64 and 0.70), Southeast Asian (AUC, 0.65), and East Asian (AUC, 0.73) ancestry. Pediatric participants with the top 5% PRS had 2.80 to 5.82 increased odds of asthma compared to the bottom 5% across the training, validation 1, and validation 2 cohorts when adjusted for ancestry. Phenome-wide association study analysis confirmed the strong association of the identified PRS with asthma (odds ratio, 2.71, PFDR = 3.71 × 10-65) and related phenotypes. CONCLUSIONS: A multiancestral PRS for asthma based on Bayesian posterior genomic effect sizes identifies increased odds of pediatric asthma.

Treatment by biomarker-informed endotype vs guideline care in children with difficult-to-treat asthma.

BACKGROUND: Asthma is heterogeneous, contributing to difficulty in disease management. OBJECTIVE: To develop a biomarker-informed treatment model for difficult-to-treat (DTT) asthma and conduct a pilot feasibility study. METHODS: School-aged children (n = 21) with DTT asthma were enrolled and completed 3 medical visits (V1-V3). V2 and V3 were completed approximately 3.5 months and 12 months after V1, respectively. At V1, guideline care and adherence interventions were initiated, and blood samples were collected for asthma biomarker assessment. A personalized treatment algorithm was developed based on biomarkers (treatment by endotype) and was implemented at V2. Asthma outcomes were compared from V1 to V2 (guideline-based care) to V2 to V3 (guideline + biomarker-informed care). RESULTS: Overall retention was 86%. There was an even distribution of participants with allergy, without allergy, and with mixed allergies. The participants received an average of 5.9 interventions (range, 3-9). The allergic phenotype was characterized by increased CDHR3 risk genotype and high transepidermal water loss. High serum interleukin-6 level was most notable in the mixed allergic subgroup. The nonallergic phenotype was characterized by vitamin D deficiency and poor steroid treatment responsiveness. The personalized treatment plans were associated with decreased emergency department visits (median, 1 vs 0; P = .04) and increased asthma control test scores (median, 22.5 vs 23.0; P = .01). CONCLUSION: The biomarker-based treatment algorithm triggered interventions on top of guideline care in all children with DTT asthma studied, supporting the need for this type of multipronged approach. Our findings identify the minimal biomarker set that is informative, reveal that this treatment-by-endotype intervention is feasible and may be superior to guideline care alone, and provide a strong foundation for a definitive trial. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT04179461.

Vitamin D, skin filaggrin, allergic sensitization, and race.

BACKGROUND: In addition to its involvement in both the innate and adaptive immune systems, vitamin D has been found to affect keratinocyte function and proliferation, suggesting a possible role for vitamin D in cutaneous allergic sensitization. OBJECTIVE: To explore the role of circulating vitamin D levels in allergic sensitization. METHODS: Serum 25-hydroxyvitamin D (25(OH)D) levels were measured in a subset of children (N = 323) enrolled in the Mechanisms of Progression of Atopic Dermatitis to Asthma in Children cohort, a prospective early life cohort of children with atopic dermatitis. Allergic sensitization was determined using skin prick testing, and FLG expression in the keratinocytes was measured by quantitative polymerase chain reaction. Multiple Poisson regression was used to evaluate interaction effects between serum 25(OH)D levels and FLG expression with sensitization load as the outcome. RESULTS: Black participants had significantly lower mean levels of serum 25(OH)D compared with non-Black participants (29.3 vs 32.9 ng/mL; P < .001). FLG expression and sensitization load were negatively correlated in non-Black participants with 25(OH)D levels less than 27.2 ng/mL (Rho = -0.45; P = .02). No association between FLG expression and sensitization load was found in Black participants or participants with 25(OH)D levels greater than or equal to 27.2 ng/mL. Multiple Poisson regression models confirmed that 25(OH)D levels interact with FLG expression to affect sensitization load in non-Black participants. CONCLUSION: Despite lower vitamin D levels in Black participants, sensitization load was associated with nonlesional skin FLG expression in non-Black, but not Black, children with low vitamin D levels. Thus, a complex interplay of factors determines the impact of vitamin D on allergic sensitization.

TSLP disease-associated genetic variants combined with airway TSLP expression influence asthma risk.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is an epithelial-derived cytokine important in initiation of allergic inflammation. Single nucleotide polymorphisms (SNPs) in TSLP are associated with asthma, yet studies have shown inconsistent associations between circulating TSLP and asthma. Studies that integrate the combined effects of TSLP genotype, TSLP mRNA, circulating TSLP levels, and asthma outcome are lacking. OBJECTIVES: This study sought to recruit a novel cohort based on asthma-relevant TSLP SNPs and determine their impact on TSLP mRNA expression and TSLP circulating protein levels, and their individual and combined effects on asthma. METHODS: This study developed an algorithm to prioritize TSLP SNPs and recruited 51 carriers and noncarriers based on TSLP genotypes. TSLP mRNA was quantified in nasal epithelial cells and circulating TSLP levels in plasma. This study determined the associations of defined TSLP risk genotypes and/or TSLP mRNA and protein levels with asthma. RESULTS: TSLP mRNA expression, but not circulating TSLP, was significantly increased in people who are asthmatic compared with in people who are nonasthmatic (P = .007; odds ratio, 1.44). Notably, 90% of children with the defined TSLP risk genotypes and high nasal TSLP mRNA expression (top tertile) had asthma compared with 40% of subjects without risk genotypes and with low TSLP expression (bottom tertile) (P = .024). No association between circulating TSLP and asthma was observed. CONCLUSIONS: Collectively, these data suggest childhood asthma is modified by the combined effects of TSLP genotype and TSLP expression in the nasal epithelium. The increased asthma risk likely manifests when genetic variation enables expression quantitative trait loci in the TSLP locus to elevate TSLP. It is important to consider both biomarkers when factoring asthma risk.

Longitudinal atopic dermatitis endotypes: An atopic march paradigm that includes Black children.

BACKGROUND: The atopic march has been studied mostly in White populations, biasing our current paradigms. OBJECTIVE: We sought to define the atopic march in Black and White children and explore mechanisms for racial differences. METHODS: Utilizing the Mechanisms of Progression of Atopic Dermatitis to Asthma in Children (MPAACH) cohort (n = 601), we assessed longitudinal sensitization, food allergy (FA), allergic rhinitis, risk of asthma development (through the Pediatric Asthma Risk Score), Scoring for Atopic Dermatitis (SCORAD), transepidermal water loss, skin filaggrin (FLG) expression, exposures, and genetic heritability to define AD progression endotypes in Black and White children. RESULTS: White MPAACH children were more likely to be sensitized to aero and food allergens (P = .0001) and over 3 times more likely to develop FA and/or allergic rhinitis (AR) without asthma risk (P < .0001). In contrast, Black children were over 6 times more likely to proceed to high asthma risk without FA, sensitization, or AR (P < .0001). White children had higher lesional and nonlesional transepidermal water loss (both P < .001) as well as decreased nonlesional keratinocyte FLG expression (P = .02). Black children had increased genetic heritability for asthma risk and higher rates of exposures to secondhand smoke and traffic-related air pollution. CONCLUSIONS: Black and White children with AD have distinct allergic trajectories defined by different longitudinal endotypes. Black children exhibit higher asthma risk despite a more intact skin barrier and less sensitization, FA, and AR. White children have less asthma risk, despite a more dysfunctional skin barrier, and more FA, AR, and sensitization. The observed racial differences are likely due in part to increased genetic heritability for asthma risk and harmful environmental exposures in Black children. Collectively, our findings provide a new paradigm for an atopic march that is inclusive of Black children.

Novel role for caspase recruitment domain family member 14 and its genetic variant rs11652075 in skin filaggrin homeostasis.

BACKGROUND: Low epidermal filaggrin (FLG) is a risk factor for atopic dermatitis (AD) and allergic comorbidity. FLG mutations do not fully explain the variation in epidermal FLG levels, highlighting that other genetic loci may also regulate FLG expression. OBJECTIVE: We sought to identify genetic loci that regulate FLG expression and elucidate their functional and mechanistic consequences. METHODS: A genome-wide association study of quantified skin FLG expression in lesional and baseline non(never)-lesional skin of children with AD in the Mechanisms of Progression of Atopic Dermatitis to Asthma in Children cohort was conducted. Clustered regularly interspaced short palindromic repeat approaches were used to create isogenic human keratinocytes differing only at the identified variant rs11652075, and caspase recruitment domain family member 14 (CARD14)-deficient keratinocytes for subsequent mechanistic studies. RESULTS: The genome-wide association study identified the CARD14 rs11652075 variant to be associated with FLG expression in non(never)-lesional skin of children with AD. Rs11652075 is a CARD14 expression quantitative trait locus in human skin and primary human keratinocytes. The T variant destroys a functional cytosine-phosphate-guanine site, resulting in reduced cytosine-phosphate-guanine methylation at this site (but not neighboring sites) in TT and CT compared with CC primary human keratinocytes and Mechanisms of Progression of Atopic Dermatitis to Asthma in Children children's skin samples, and rs11652075 increases CARD14 expression in an allele-specific fashion. Furthermore, studies in clustered regularly interspaced short palindromic repeat-generated CC and TT isogenic keratinocytes, as well as CARD14-haplosufficient and deficient keratinocytes, reveal that IL-17A regulates FLG expression via CARD14, and that the underlying mechanisms are dependent on the rs11652075 genotype. CONCLUSIONS: Our study identifies CARD14 as a novel regulator of FLG expression in the skin of children with AD. Furthermore, CARD14 regulates skin FLG homeostasis in an rs11652075-dependent fashion.