Carbonic anhydrases in human keratinocytes and their regulation by all-trans retinoic acid and 1α,25-dihydroxyvitamin D3.

Carbonic anhydrases (CAs) are ubiquitously expressed enzymes and catalyse an important physiological reaction of interconverting the hydration of carbon dioxide to bicarbonates, which is crucial for maintaining acid/base equilibrium in certain tissues. While 15 different isoforms of CAs are present in various cell types in human tissues, their expression pattern in the epidermis remains to be investigated. Here, we report the expression of 5 CA isoforms (CAII, CAIX, CAXI, CAXII and CAXIII) in human primary keratinocytes. Further, we demonstrate that the expression of CAII and CAIX in these cells is significantly up-regulated by the biologically active metabolites of vitamin A (all-trans retinoic acid) and vitamin D (1α,25-dihydroxyvitamin D3 ), respectively. Taken together, apart from providing new information on the expression of CAs in the skin, our results highlight a previously undisclosed connection between vitamin A and CAII expression and vitamin D and CAIX expression.

Different phenotypes and factors associated with atopic dermatitis in the young adult Singaporean Chinese population: A cross-sectional study.

BACKGROUND: Atopic dermatitis (AD) is a chronic allergic disease typically accompanied by atopy and thus, a tendency to develop allergic diseases such as allergic rhinitis, asthma or food allergies. Currently, individuals with AD are classified into those presenting with AD alone and those presenting with AD along with other allergic diseases (AD+). It is important to identify the various endophenotypes of AD using anthropometric, environmental, socio-economic, and disease history data in order to improve disease management. To characterize the phenotypic differences among Singaporean Chinese individuals with AD alone and AD+, and identify the socioeconomic, lifestyle, and environmental factors associated with these different presentations. METHODS: Based on data collected via a standardized/validated questionnaire, 4604 participants (mean age: 22.1 years) were classified into three groups: 1) AD alone group; 2) AD with other allergic diseases group (AD+); and 3) Control group. RESULTS: Participants were less sensitized to common inhalant allergens in the AD alone group versus the Control group (67% vs. 72%, respectively; p < 0.05).High Body Mass Index (i.e., BMI > 23) was associated with the disease and the difference was more pronounced in the AD alone group compared to the AD+ group (Odds Ratio: 1.38; 95% Confidence Interval: 1.4-1.67; p < 0.001). No major differences in habits were observed between the AD alone and AD+ groups. CONCLUSIONS: The two presentations of AD may have different underlying pathogenesis and associated risk factors.

Toll-like Receptor 3 Agonist, Polyinosinic-polycytidylic Acid, Upregulates Carbonic Anhydrase II in Human Keratinocytes.

Carbonic anhydrases are ubiquitously expressed enzymes that reversibly hydrate carbon dioxide to bicarbonate and protons. While the main function of carbonic anhydrases is to regulate pH and osmotic balance, their involvement in other physiological processes remains to be explored. This study analysed changes in mRNA and protein levels of carbonic anhydrase II in human primary keratinocytes treated with various toll-like receptor agonists and cytokines. A significant upregulation of carbonic anhydrase II at the mRNA and protein levels was observed upon treatment with polyinosinic-polycytidylic acid, a toll-like receptor 3 agonist. Furthermore, in agreement with the increased expression of carbonic anhydrase II in atopic dermatitis skin, carbonic anhydrase II was upregulated by the Th2 cytokines interleukins -4 and -13. In conclusion, these results suggest a potential role of carbonic anhydrase II in Th2-dependent and toll-like receptor 3-induced pathways in inflammatory skin conditions.

Whole metagenome profiling reveals skin microbiome-dependent susceptibility to atopic dermatitis flare.

Whole metagenome analysis has the potential to reveal functional triggers of skin diseases, but issues of cost, robustness and sampling efficacy have limited its application. Here, we have established an alternative, clinically practical and robust metagenomic analysis protocol and applied it to 80 skin microbiome samples epidemiologically stratified for atopic dermatitis (AD). We have identified distinct non-flare, baseline skin microbiome signatures enriched for Streptococcus and Gemella but depleted for Dermacoccus in AD-prone versus normal healthy skin. Bacterial challenge assays using keratinocytes and monocyte-derived dendritic cells established distinct IL-1-mediated, innate and Th1-mediated adaptive immune responses with Staphylococcus aureus and Staphylococcus epidermidis. Bacterial differences were complemented by perturbations in the eukaryotic community and functional shifts in the microbiome-wide gene repertoire, which could exacerbate a dry and alkaline phenotype primed for pathogen growth and inflammation in AD-susceptible skin. These findings provide insights into how the skin microbial community, skin surface microenvironment and immune system cross-modulate each other, escalating the destructive feedback cycle between them that leads to AD flare.

Functional variants of 17q12-21 are associated with allergic asthma but not allergic rhinitis.

BACKGROUND: Allergic rhinitis (AR) and asthma are common allergic conditions with a shared genetic component to their cause. The 17q12-21 locus includes several genes that have been linked to asthma susceptibility, but the role of this locus in AR is unclear. Asthma and AR in adults of Chinese ethnicity in Singapore are predominately caused by sensitization against house dust mites with a nearly complete penetrance of the allergen, which presents a unique opportunity for accurately identifying genetic associations with allergic diseases. OBJECTIVE: We sought to define the functional role of 17q12-21 in patients with AR and allergic asthma. METHODS: We asked whether single nucleotide polymorphisms (SNPs) in the 17q12-21 locus were associated with AR or asthma in a cohort of 3460 ethnic Chinese subjects residing in Singapore (1435 in the discovery phase and 2025 in the validation phase). Full-blood mRNA gene expression data, plasma IgE levels, and immune cell frequencies in peripheral blood were tested against the tag SNP genotypes. Luciferase assays were used to measure the effect of putative promoter SNPs on expression of the asthma-associated orosomucoid-like 3 gene (ORMDL3). RESULTS: Within 17q12-21, only the tag SNP rs8076131 was significantly associated with asthma (P = 8.53 × 10(-10); odds ratio, 0.6715), and AR status was independent of SNPs in this region. C-A alleles at rs8076131 resulted in significantly increased ORMDL3 expression in HEK293 cells in vitro relative to T-G alleles. Moreover, subjects with the risk genotype AA exhibited significantly higher total IgE levels and higher blood eosinophil counts than those with the lower-risk genotypes. CONCLUSION: The 17q12-21 locus has a strong genetic association with allergic asthma but not with AR. The polymorphic effect of this locus is attributed to the linkage set tagged by rs8076131, which affects the expression of ORMDL3, protein phosphatase 1, regulatory inhibitor subunit 1B (PPP1R1B), zona pellucida binding protein 2 (ZPBP2), and gasdermin B (GSDMB) and is correlated with high IgE levels and eosinophil counts in subjects bearing the risk genotype.

A functional brain-derived neurotrophic factor (BDNF) gene variant increases the risk of moderate-to-severe allergic rhinitis.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is a secretory protein that has been implicated in the pathogenesis of allergic rhinitis (AR), atopic asthma, and eczema, but it is currently unknown whether BDNF polymorphisms influence susceptibility to moderate-to-severe AR. OBJECTIVE: We sought to identify disease associations and the functional effect of BDNF genetic variants in patients with moderate-to-severe AR. METHODS: Tagging single nucleotide polymorphisms (SNPs) spanning the BDNF gene were selected from the human HapMap Han Chinese from Beijing (CHB) data set, and associations with moderate-to-severe AR were assessed in 2 independent cohorts of Chinese patients (2216 from Shandong province and 1239 living in Singapore). The functional effects of the BDNF genetic variants were determined by using both in vitro and ex vivo assays. RESULTS: The tagging SNP rs10767664 was significantly associated with the risk of moderate-to-severe AR in both Singapore Chinese (P = .0017; odds ratio, 1.324) and Shandong Chinese populations (P = .039; odds ratio, 1.180). The coding nonsynonymous SNP rs6265 was in perfect linkage with rs10767664 and conferred increased BDNF protein secretion by a human cell line in vitro. Subjects bearing the AA genotype of rs10767664 exhibited increased risk of moderate-to-severe AR and displayed increased BDNF protein and total IgE levels in plasma. Using a large-scale expression quantitative trait locus study, we demonstrated that BDNF SNPs are significantly associated with altered BDNF concentrations in peripheral blood. CONCLUSION: A common genetic variant of the BDNF gene is associated with increased risk of moderate-to-severe AR, and the AA genotype is associated with increased BDNF mRNA levels in peripheral blood. Together, these data indicate that functional BDNF gene variants increase the risk of moderate-to-severe AR.

Genome-wide association study identifies PERLD1 as asthma candidate gene.

BACKGROUND: Recent genome-wide association studies (GWAS) for asthma have been successful in identifying novel associations which have been well replicated. The aim of this study is to identify the genetic variants that influence predisposition towards asthma in an ethnic Chinese population in Singapore using a GWAS approach. METHODS: A two-stage GWAS was performed in case samples with allergic asthma, and in control samples without asthma and atopy. In the discovery stage, 490 case and 490 control samples were analysed by pooled genotyping. Significant associations from the first stage were evaluated in a replication cohort of 521 case and 524 control samples in the second stage. The same 980 samples used in the discovery phase were also individually genotyped for purposes of a combined analysis. An additional 1445 non-asthmatic atopic control samples were also genotyped. RESULTS: 19 promising SNPs which passed our genome-wide P value threshold of 5.52 × 10-8 were individually genotyped. In the combined analysis of 1011 case and 1014 control samples, SNP rs2941504 in PERLD1 on chromosome 17q12 was found to be significantly associated with asthma at the genotypic level (P = 1.48 × 10-6, ORAG = 0.526 (0.369-0.700), ORAA = 0.480 (0.361-0.639)) and at the allelic level (P = 9.56 × 10-6, OR = 0.745 (0.654-0.848)). These findings were found to be replicated in 3 other asthma GWAS studies, thus validating our own results. Analysis against the atopy control samples suggested that the SNP was associated with allergic asthma and not to either the asthma or allergy components. Genotyping of additional SNPs in 100 kb flanking rs2941504 further confirmed that the association was indeed to PERLD1. PERLD1 is involved in the modification of the glycosylphosphatidylinositol anchors for cell surface markers such as CD48 and CD59 which are known to play multiple roles in T-cell activation and proliferation. CONCLUSIONS: These findings reveal the association of a PERLD1 as a novel asthma candidate gene and reinforce the involvement of genes on the 17q12-21 chromosomal region in the etiology of asthma.

Genetic variation in BDNF is associated with allergic asthma and allergic rhinitis in an ethnic Chinese population in Singapore.

Allergic diseases affect more than 25% of the world population and result from a complex interplay between genetic and environmental factors. Recent evidence has shown that BDNF (Brain Derived Neurotrophic Factor) could serve as an important marker of allergic disease. Increased levels of BDNF in blood, bronchoalveolar lavage fluid and nasal lavage fluid positively correlate with disease activity and severity in patients with allergic rhinitis (AR), asthma and atopic eczema. However, reports on the association between genetic variation in BDNF and allergic disease have been controversial. This study therefore aims to clarify the relationship between single nucleotide polymorphisms (SNPs) in BDNF and a genetic predisposition to AR and asthma in an ethnic Chinese population of Singapore. Volunteers with a self-reported history of asthma (718 subjects) or a history of AR as determined by a researcher-administered questionnaire (795 subjects) were used in this study, alongside controls with no personal or family history of allergy (717 subjects). The association results identified a significant association for the tagSNP rs10767664 with a significant PDominant=0.0007 and OR=1.3 for AR and PDominant=0.0005 and OR=1.3 for asthma (using a dominant model of association). The haplotype based analysis also identified a significant association further confirming the single SNP association. The SNP rs10767664 is strongly linked (r2=0.95) to the functional polymorphism rs6265 (Val66Met), which has previously been reported to be associated to allergic phenotypes and also shown to affect BDNF expression. BDNF is a therefore a key molecular player in allergy. Further studies on polymorphisms within BDNF may shed light on its role in the pathogenesis of allergic diseases and potentially serve as biomarkers for allergic disease.

Genome-wide association study for atopy and allergic rhinitis in a Singapore Chinese population.

Allergic rhinitis (AR) is an atopic disease which affects about 600 million people worldwide and results from a complex interplay between genetic and environmental factors. However genetic association studies on known candidate genes yielded variable results. The aim of this study is to identify the genetic variants that influence predisposition towards allergic rhinitis in an ethnic Chinese population in Singapore using a genome-wide association study (GWAS) approach. A total of 4461 ethnic Chinese volunteers were recruited in Singapore and classified according to their allergic disease status. The GWAS included a discovery stage comparing 515 atopic cases (including 456 AR cases) and 486 non-allergic non-rhinitis (NANR) controls. The top SNPs were then validated in a replication cohort consisting of a separate 2323 atopic cases (including 676 AR cases) and 511 NANR controls. Two SNPs showed consistent association in both discovery and replication phases; MRPL4 SNP rs8111930 on 19q13.2 (OR = 0.69, P(combined) = 4.46×10(-05)) and BCAP SNP rs505010 on chromosome 10q24.1 (OR = 0.64, P(combined) = 1.10×10(-04)). In addition, we also replicated multiple associations within known candidates regions such as HLA-DQ and NPSR1 locus in the discovery phase. Our study suggests that MRPL4 and BCAP, key components of the HIF-1α and PI3K/Akt signaling pathways respectively, are two novel candidate genes for atopy and allergic rhinitis. Further study on these molecules and their signaling pathways would help in understanding of the pathogenesis of allergic rhinitis and identification of targets for new therapeutic intervention.

Variation in Uteroglobin-Related Protein 1 (UGRP1) gene is associated with allergic rhinitis in Singapore Chinese.

BACKGROUND: Uteroglobin-Related Protein 1 (UGRP1) is a secretoglobulin protein which has been suggested to play a role in lung inflammation and allergic diseases. UGRP1 has also been shown to be an important pneumoprotein, with diagnostic potential as a biomarker of lung damage. Previous genetic studies evaluating the association between variations on UGRP1 and allergic phenotypes have yielded mixed results. The aim of this present study was to identify genetic polymorphisms in UGRP1 and investigate if they were associated with asthma and allergic rhinitis in the Singapore Chinese population. METHODS: Resequencing of the UGRP1 gene was conducted on 40 randomly selected individuals from Singapore of ethnic Chinese origin. The polymorphisms identified were then tagged and genotyped in a population of 1893 Singapore Chinese individuals. Genetic associations were evaluated in this population comparing 795 individuals with allergic rhinitis, 718 with asthma (of which 337 had both asthma and allergic rhinitis) and 717 healthy controls with no history of allergy or allergic diseases. RESULTS: By resequencing the UGRP1 gene within our population, we identified 11 novel and 16 known single nucleotide polymorphisms (SNPs). TagSNPs were then genotyped, revealing a significant association between rs7726552 and allergic rhinitis (Odds Ratio: 0.81, 95% Confidence Interval: 0.66-0.98, P = 0.039). This association remained statistically significant when it was analyzed genotypically or when stratified according to haplotypes. When variations on UGRP1 were evaluated against asthma, no association was observed. CONCLUSION: This study documents the association between polymorphisms in UGRP1 and allergic rhinitis, suggesting a potential role in its pathogenesis.