Childhood asthma is associated with mutations and gene expression differences of ORMDL genes that can interact.

BACKGROUND: Genomewide association studies identified ORMDL3 as a plausible asthma candidate gene. ORMDL proteins regulate sphingolipid metabolism and ceramide homeostasis and participate in lymphocyte activation and eosinophil recruitment. Strong sequence homology between the three ORMDL genes and ORMDL protein conservation among different species suggest that they may have shared functions. We hypothesized that if single nucleotide polymorphisms (SNPs) in ORMDL3 alter its gene expression and play a role in asthma, variants in ORMDL1 and ORMDL2 might also be associated with asthma. METHODS: Asthma associations of 44 genotyped SNPs were determined in at least 1303 subjects (651 asthmatics). ORMDL expression was evaluated in peripheral blood mononuclear cells (PBMC) from 55 subjects (eight asthmatics) before and after allergen stimulation, and in blood (n = 60, 5 asthmatics). Allele-specific cis-effects on ORMDL expression were assessed. Interactions between human ORMDL proteins were determined in living cells. RESULTS: Sixteen SNPs in all three ORMDLs were associated with asthma (14 in ORMDL3). Baseline expression of ORMDL1 (P = 1.7 × 10(-6) ) and ORMDL2 (P = 4.9 × 10(-5) ) was significantly higher in PBMC from asthmatics, while induction of ORMDLs upon stimulation was stronger in nonasthmatics. Disease-associated alleles (rs8079416, rs4795405, rs3902920) alter ORMDL3 expression. ORMDL proteins formed homo- and heterooligomers and displayed similar patterns of interaction with SERCA2 and SPT1. CONCLUSIONS: Polymorphisms in ORMDL genes are associated with asthma. Asthmatics exhibit increased ORMDL levels, suggesting that ORMDLs contribute to asthma. Formation of heterooligomers and similar interaction patterns with proteins involved in calcium homeostasis and sphingolipid metabolism could indicate shared biological roles of ORMDLs, influencing airway remodeling and hyperresponsiveness.

Fine-mapping of IgE-associated loci 1q23, 5q31, and 12q13 using 1000 Genomes Project data.

BACKGROUND: Genome-wide association studies (GWAS) repeatedly identified 1q23 (FCER1A), 5q31 (RAD50-IL13 and IL4), and 12q13 (STAT6) as major susceptibility loci influencing the regulation of total serum IgE levels. As GWAS may be insufficient to capture causal variants, we performed fine-mapping and re-genotyping of the three loci using 1000 Genomes Project datasets. METHODS: Linkage disequilibrium tagging polymorphisms and polymorphisms of putative functional relevance were genotyped by chip technology (24 polymorphisms) or MALDI-TOF-MS (40 polymorphisms) in at least 1303 German children (651 asthmatics). The effect of polymorphisms on total serum IgE, IgE percentiles, and atopic diseases was assessed, and a risk score model was applied for gene-by-gene interaction analyses. Functional effects of putative causal variants from these three loci were studied in silico. RESULTS: Associations from GWAS were confirmed and extended. For 1q23 and 5q31, the majority of associations were found with mild to moderately elevated IgE levels, while in the 12q13 locus, single-nucleotide polymorphisms (SNPs) were associated with strongly elevated IgE levels. Gene-by-gene interaction analyses suggested that the presence of mutations in all three loci increases the risk for elevated IgE up to fourfold. CONCLUSION: This fine-mapping study confirmed previous associations and identified novel associations of SNPs in 1q23, 5q31, and 12q13 with different levels of serum IgE and their concomitant contribution to IgE regulation.

A polymorphism in the TH 2 locus control region is associated with changes in DNA methylation and gene expression.

BACKGROUND: Genomewide association and epigenetic studies found a region within the RAD50 gene on chromosome 5q31 to be associated with total serum IgE levels and asthma. In mice, this region harbors a locus control region for nearby TH 2 cytokines, which is characterized by four Rad50 DNase I hypersensitive sites (RHS4-7). Among these, RHS7 seems to have the strongest impact on TH 2 differentiation. We investigated whether within the human homolog of RHS7, functional polymorphisms exist, which could affect DNA methylation or gene expression in the 5q31 locus and might have an influence on asthma status or IgE regulation. METHODS: The human RHS7 region was fine mapped using 1000 genomes database information. In silico analysis and electrophoretic mobility shift assays were used to assess SNP function. Allele-specific effects on DNA methylation were evaluated in cord blood (n = 73) and at age of 4.5 years (n = 61) by pyrosequencing. Allele-specific effects on RAD50, IL4, and IL13 expression were analyzed in 100 subjects. Associations with asthma and IgE levels were investigated in the MAGICS/ISAAC II population (n = 1145). RESULTS: Polymorphism rs2240032 in the RHS7 region is suggestive of allele-specific transcription factor binding, affects methylation of the IL13 promoter region and influences RAD50 and IL4 expression (lowest P = 0.0027). It is also associated with total serum IgE levels (P = 0.0227). CONCLUSION: A functional relevant polymorphism in the TH 2 locus control region, equivalent to RHS7 in mice, affects DNA methylation and gene expression within 5q31 and influences total serum IgE on the population level.

A role of FCER1A and FCER2 polymorphisms in IgE regulation.

BACKGROUND: Both FCER2 and FCER1A encode subunits of IgE receptors. Variants in FCER1A were previously identified as major determinants of IgE levels in genome-wide association studies. METHODS: Here we investigated in detail whether FCER2 polymorphisms affect IgE levels alone and/or by interaction with FCER1A polymorphisms. To cover the genetic information of FCER2, 21 single-nucleotide polymorphisms (SNPs) were genotyped by Illumina HumanHap300 BeadChip (5 SNPs) and the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; 14 SNPs) in at least 1303 Caucasian children (651 asthmatics) (ISAAC II/ MAGICS population); genotypes of two SNPs were imputed. RESULTS: SNP rs3760687 showed the most consistent effect on total serum IgE levels (b [SE] = -0.38 [0.16]; P = 0.016), while FCER2 polymorphisms in general were predominantly associated with mildly-to-moderately increased IgE levels (50th and 66th percentiles). Gene-by-gene interaction analysis suggests that FCER2 polymorphism rs3760687 influences IgE levels mainly in individuals not homozygous for the risk allele of FCER1A polymorphism rs2427837, which belongs to the major IgE-determining tagging bin in the population. CONCLUSION: FCER2 polymorphism rs3760687 affects moderately elevated total serum IgE levels, especially in the absence of homozygosity for the risk allele of FCER1A SNP rs2427837.

Antibodies to N-homocysteinylated albumin and haemoglobin in patients with rheumatoid arthritis: a potential new marker of disease severity.

OBJECTIVES: To investigate whether anti-Nε-homocysteinylated albumin (anti-N-Hcy-Alb) and haemoglobin (anti-N-Hcy-Hb) antibodies occur in rheumatoid arthritis (RA) and whether they are associated with RA activity and/or severity. METHOD: Plasma total homocysteine (tHcy) and serum anti-N-Hcy-Alb and -Hb antibodies levels were determined in 76 RA patients (12 men and 64 women, median age 56 years) and 80 age- and sex-matched controls. RESULTS: RA patients compared to healthy controls demonstrated elevated tHcy [median (IQR), 13.20 (3.80) vs. 9.45 (3.25) µmol/L; p < 0.000001] and anti-N-Hcy-Alb and -Hb antibodies [absorbance at 490 nm, median (IQR), 0.546 (0.085) vs. 0.452 (0.056) and 0.649 (0.106) vs. 0.532 (0.057), respectively; all p < 0.000001]. In RA patients, RA radiological class was a strong independent predictor of tHcy [ß (SE), 0.59 (0.11); p = 0.000001] and anti-N-Hcy-Alb [0.36 (0.12); p = 0.003] and -Hb [0.49 (0.11); p = 0.00007] antibodies. The number of swollen joints, but not C-reactive protein (CRP), interleukin 6 (IL-6), positive rheumatoid factor (RF), or anti-cyclic citrullinated peptide (anti-CCP) antibodies, showed independent effects on anti-N-Hcy-Alb [ß (SE), 0.36 (0.11); p = 0.001] and -Hb [0.25 (0.11); p = 0.02] antibodies. Anti-N-Hcy-Hb antibodies, but not those against N-Hcy-Alb, were positively correlated with RA functional class and RA duration. No effect of any medications on tHcy or anti-N-Hcy-protein antibodies was observed. CONCLUSIONS: This study is the first to show that RA is characterized by enhanced autoimmune response to Nε-homocysteinylated proteins detectable in circulating blood, which is related to some clinical measures of RA severity.

Current concepts of IgE regulation and impact of genetic determinants.

Immunoglobulin E (IgE) mediated immune responses seem to be directed against parasites and neoplasms, but are best known for their involvement in allergies. The IgE network is tightly controlled at different levels as outlined in this review. Genetic determinants were suspected to influence IgE regulation and IgE levels considerably for many years. Linkage and candidate gene studies suggested a number of loci and genes to correlate with total serum IgE levels, and recently genome-wide association studies (GWAS) provided the power to identify genetic determinants for total serum IgE levels: 1q23 (FCER1A), 5q31 (RAD50, IL13, IL4), 12q13 (STAT6), 6p21.3 (HLA-DRB1) and 16p12 (IL4R, IL21R). In this review, we analyse the potential role of these GWAS hits in the IgE network and suggest mechanisms of how genes and genetic variants in these loci may influence IgE regulation.

An association of TLR2­16934A >T polymorphism and severity/phenotype of atopic dermatitis.

BACKGROUND: Toll-like receptor 2 gene (TLR2) ­16934A>T polymorphism has been shown to be associated with severity of atopic dermatitis (AD) as measured using severity scoring of atopic dermatitis (SCORAD) index. Moreover, TLR2­16934A>T polymorphism has been associated with atopy and allergic disorders in farmers' children. OBJECTIVE: The aim of this study was to evaluate an association between TLR2­16934A>T polymorphism and AD phenotype, including disease severity and concomitant atopic diseases, or potential serum markers of AD severity and also to find a molecular background of the clinical associations. METHODS: Genotyping for TLR2­16934A>T polymorphism was performed in 130 consecutive adult ambulatory patients with AD. Total serum (TS) IgE levels, serum tryptase, plasma interleukin-6 and C-reactive protein were measured. In addition, luciferase assay and electrophoretic-mobility shift assay were conducted to assess the effect of ­16934A>T polymorphism on transcriptional activity. RESULTS: There was an inverse association of TLR2­16934TT genotype and/or ­16934T allele with SCORAD, but not with TS IgE, tryptase or inflammatory markers. Interestingly, ­16934AA genotype and/or ­16934A allele were overrepresented in AD patients with concomitant asthma or a family history of atopy. In a subgroup analysis, TLR2­16934A>T polymorphism was associated with SCORAD, asthma, allergic conjunctivitis or family history of atopy in AD patients with TS IgE ≥106 IU/mL but not in those having TS IgE <106 IU/mL. Functional analyses showed that TLR2­16934T allele is associated with higher luciferase activity in human monocytic THP-1 cells and preferential binding of the THP-1-derived nuclear protein. CONCLUSION: TLR2­16934A>T polymorphism could be a genetic predictor of AD severity, the coexistence of asthma or atopic conjunctivitis as well as a family history of atopic diseases, especially in subjects having higher TS IgE. TLR2­16934A>T polymorphism affects transcriptional activity, which may at least in part account for the clinical associations observed for the ­16934A>T polymorphism.

FcepsilonRIalpha gene (FCER1A) promoter polymorphisms and total serum IgE levels in Japanese atopic dermatitis patients.

Two promoter polymorphisms of the high-affinity IgE receptor alpha-subunit (FcepsilonRIalpha) gene (FCER1A), -66T>C (rs2251746) and -315C>T (rs2427827), were analysed in Japanese atopic dermatitis subjects. Patients with the -315CT/TT genotype tended to have higher total serum IgE levels, while the proportion of -315CT/TT genotype or the -315T allele was significantly higher in those with highly elevated total serum IgE concentrations.

Interaction of functional FCER2 promoter polymorphism and phenotype-associated haplotypes.

Low-affinity IgE receptor gene (FCER2) rs3760687 polymorphism was found to be associated with differential binding affinity of transcription factors Sp1 and Sp3 leading to altered transcriptional activity. Haplotypic interaction of functional FCER2 polymorphisms (rs28364072, rs2228137 and rs3760687) might potentially provide a background for genotype-phenotype associations previously observed for some rather non-functional FCER2 variants.

FCER1A gene proximal promoter polymorphisms in Caucasians and East Asians.

Two FCER1A gene proximal promoter polymorphisms, -344C > T and -95T > C, both associated with total serum IgE and/or allergic disorders in Caucasians and East Asians, were shown to influence the gene expression in additive manner. In face of the rarity of other proximal promoter variants in Caucasians or their lack in East Asians, future investigations of the FCER1A locus in these ethnic groups should probably focus on three common haplotypes of the common -344C > T and -95T > C polymorphisms.

Association between inflammatory markers and the interleukin-6 -174 G/C polymorphism is abolished in peripheral arterial occlusive disease.

AIM: The interleukin-6 (IL-6) -174 G/C polymorphism has been reported to determine IL-6 levels and contribute to the development of cardiovascular disorders. The aim of our study was to evaluate the effect of the IL-6 -174 G/C polymorphism on hemostatic or inflammatory markers in patients with peripheral arterial occlusive disease (PAOD), a common manifestation of obliterative atherosclerosis. METHODS: Plasma IL-6, fibrinogen, C-reactive protein (CRP), tissue plasminogen activator, plasminogen activator inhibitor-1 (PAI-1), fibrinopeptide A and intercellular adhesion molecule-1 levels were determined in PAOD patients (n=50) and healthy controls (n=30) genotyped for the IL-6 -174 G/C polymorphism. RESULTS: In the control group, IL-6, CRP and fibrinogen levels were significantly associated with the IL-6 -174 G/C polymorphism with a gene-dosage effect being the highest in the CC subjects and the lowest in those with the GG genotype (P<0.0001, P=0.0002 and P=0.0001, respectively). Interestingly, the CC homozygotes had lower PAI-1 levels than carriers of the G allele (P=0.04). In PAOD patients, the IL-6 -174 G/C polymorphism had no effect on all the variables measured. CONCLUSION: In contrast to apparently healthy subjects, the IL-6 -174 G/C polymorphism showed no association with plasma IL-6, CRP, fibrinogen and PAI-1 levels in PAOD patients.

Assessment of Brain Derived Neurotrophic Factor in hair to study stress responses: A pilot investigation.

To study pathogenic stress-effects in health and disease, it is paramount to define easy access parameters for non-invasive analysis of biological change in response to stress. Hair samples successfully provide this access for the study of hypothalamus-pituitary-adrenal axis (HPA) changes. In this study, we assess the hair expression and corresponding epigenetic changes of a neurotrophin essential for autonomic nervous system function and mental health: brain derived neurotrophic factor (BDNF). In three independent studies in healthy academic volunteers (study I: German students, N=36; study II, German academic population sample, N=28; study III: Mexican students, N=115), BDNF protein expression or BDNF gene (BDNF) histone acetylation was determined. Simultaneously, mental distress and distress-associated somatic complaints were assessed by self-report. In study I, we found a negative correlation between hair-BDNF protein level and hair-cortisol as well as between hair-BDNF and somatic complaints, while hair-cortisol correlated positively with mental distress. In study II, we found a negative correlation between H4 histone acetylation at the BDNF gene P4-promoter and somatic complaints. Regression analysis confirmed confounder stability of associations in both studies. In study III, we confirmed study I and found lower hair-BDNF protein level in volunteers with high somatic complaints, who also reported higher mental distress during the end of term exams. The results indicate that BDNF protein levels can be detected in clipped hair and are associated with somatic complaints and stress in life. In addition, we concluded that plucked hair can provide material for the study of epigenetic changes in stress-affected tissues. These tools can prove valuable for future studies on distress, both under experimental and field conditions.

Atorvastatin and quinapril inhibit blood coagulation in patients with coronary artery disease following 28 days of therapy.

BACKGROUND: We evaluated the antithrombotic effects of statins and angiotensin-converting enzyme inhibitor (ACEI) drugs in patients with coronary artery disease (CAD). METHODS AND RESULTS: Blood coagulation at the site of microvascular injury was assessed in 26 males with CAD before and after treatment with quinapril (10 mg day-1; n=13) or atorvastatin (40 mg day-1; n=13) for 4 weeks and an additional 4 weeks of combined therapy (quinapril+atorvastatin). Rates of prothrombin and factor V activation (FVa), fibrinogen (Fbg) cleavage and FVa inactivation showed that both quinapril and atorvastatin decreased the rates of: formation of thrombin B-chain (by 30.6%, P=0.007; and by 34.3%, P=0.003), formation of thrombin-antithrombin complexes (by 30.4%, P=0.0002; and by 40%, P=0.001), FV activation (by 19.1%, P=0.03; and by 21.8%, P=0.005) and Fbg depletion (by 29.2%, P=0.004; and by 32.7%, P=0.001). Atorvastatin alone accelerated FVa inactivation (P=0.005). A further 4 weeks of combined therapy enhanced most anticoagulant effects only when atorvastatin was added to quinapril. CONCLUSIONS: In CAD patients, atorvastatin and quinapril slowed blood clotting at the site of microvascular injury after 28 days of therapy. Addition of atorvastatin to quinapril, but not quinapril to the statin, enhanced the anticoagulant effects. Our findings might help explain the reduced risk of myocardial infarction or stroke in patients treated with statins and/or ACEIs and the lack of clinical benefits from ACEI added to prior statin therapy in patients at cardiovascular risk.

The alpha-chain of high-affinity receptor for IgE (FcepsilonRIalpha) gene polymorphisms and serum IgE levels.

BACKGROUND: The high-affinity receptor for immunoglobulin-E (IgE) (FcepsilonRI) plays a major role in the pathogenesis of allergy, but there are only two published studies on its alpha subunit (FcepsilonRIalpha) genetic variability in allergic diseases. AIMS OF THE STUDY: Mutational screening in the region of the FcepsilonRIalpha gene promoter and the first exon with subsequent genetic variability assessment in allergic patients and a random population sample. METHODS: Allergic subjects were individuals with asthma or urticaria. Age- and sex-matched controls were randomly selected from a large population sample. Mutational screening was performed using a single-stranded conformational polymorphism and subsequent sequencing. Detected polymorphisms were genotyped by restriction fragment length polymorphism. Total serum IgE was measured in allergic subjects and controls. Skin prick tests, blood eosinophil count and aspirin challenge test were performed only in the subjects. A subgroup of the subjects was further characterized by autologous serum skin test, histamine release test, Phadiatop and IgE antibodies against staphylococcal enterotoxins. RESULTS: Two linked polymorphisms -344 C>T and -95 T>C were found within the FcepsilonRIalpha gene. The allele -344 T frequency was 0.45 vs 0.37 (P = 0.33), and the allele -95 C frequency was 0.26 in subjects vs 0.30 in controls (P = 0.62). Serum IgE was significantly higher in subjects homozygous for the -344T allele (TT genotype) than in those carrying the -344 C allele (CT or CC genotype; P = 0.003), but this association was not detectable in controls. CONCLUSIONS: Our findings of genotype-related differences in IgE levels in allergic patients suggest an impact of -344 C>T but not -95 T>C gene polymorphism of FcepsilonRIalpha on total levels of IgE. The genetic variability in FcepsilonRIalpha at the -344 nucleotide of its regulatory sequence, though not related to atopy, predicts higher levels of the immunoglobulin.