María Isidoro-García

Epigenetic changes in B lymphocytes associated with house dust mite allergic asthma.

Although there is no doubt about the influence of the genetic background in the onset of the allergic diseases, Epigenome-Wide Association Studies are needed to elucidate the possible relationship between allergic diseases and epigenomic dysregulation. In this study we aimed to analyze the epigenetic patterns, in terms of DNA methylation, of three well-characterized populations of house dust mite allergic subjects, aspirin-intolerant asthmatics and controls. As a first, genome-wide phase, we used the HELP assay to study the methylation patterns in CD19+ B lymphocytes in these populations, and found that there are reproducible epigenetic differences at limited numbers of loci distinguishing the groups, corroborated by bisulphite MassArray in a second validation phase of an expanded 40 subject group. These validated epigenetic changes occur at loci characterized as important for the immune response. One such locus is a new candidate gene, CYP26A1, which shows differential methylation patterns and expression levels between groups. Our results suggest that epigenomic dysregulation may contribute to the susceptibility to allergic diseases, showing for the first time differences in DNA methylation between allergic and non-allergic healthy subjects, both globally and at specific loci. These observations indicate that the epigenome may offer new pathophysiological insights and therapeutic targets in atopic diseases.

Interleukin-4 (IL4) and Interleukin-4 receptor (IL4RA) polymorphisms in asthma: a case control study

BackgroundIL4/IL4RA pathway plays an important role in atopy and asthma. Different polymorphisms in IL4 and IL4RA genes have been described. Particularly, -33C>TIL4 and 576Q>RIL4RA SNPs have been independently associated to atopy and asthma. The purpose of this study was to analyse these polymorphisms in a population of patients with a well-characterized asthma phenotype.MethodsA total of 212 unrelated Caucasian individuals, 133 patients with asthma and 79 healthy subjects without symptoms or history of asthma or atopy and with negative skin prick tests were recruited. Lung function was measured by spirometry and asthma was specialist physician-diagnosed according to the ATS (American Thoracic Society) criteria and classified following the GINA (Global Initiative for Asthma) guidelines. Skin prick tests were performed according to EAACI recommendations. -33C>TIL4 was studied with TaqMan assay and 576Q>RIL4RA by PCR-RFLP technique. Hardy-Weinberg equilibrium was analysed in all groups. Dichotomous variables were analysed using χ2, Fisher exact test, Monte Carlo simulation test and odds ratio test. To model the effects of multiple covariates logistic regression was used.ResultsNo statistically significant differences between the group of patients with asthma and the controls were found when the allele and genotype distribution of -33C>TIL4 and 576Q>RIL4RA polymorphisms were compared. However, the T allele of the -33C>TIL4 SNP was more frequent in patients with persistent asthma. Multivariate analysis adjusted for age and sex confirmed that carriers of allele T had an increased risk of persistent asthma (OR:2.77, 95%CI:1.18–6.49; p = 0.019). Analysis of combination of polymorphisms showed that patients carrying both the T allele of -33C>TIL4 and the A allele of 576Q>RIL4RA had an increased risk of asthma. This association was particularly observed in persistent asthma [Fishers p value = 0.0021, Monte Carlo p value (after 104 simulations) = 0.0016, OR:3.39; 95% CI:1.50–7.66].ConclusionOur results show a trend of association between the genetic combination of the T allele of -33C>TIL4 and the A allele of 576Q>RIL4RA with asthma. This genetic variant was more frequently observed in patients with persistent asthma. As long as this study was performed in a small population, further studies in other populations are needed to confirm these results.

PTGDR gene in asthma: a functional, genetic, and epigenetic study.

To cite this article: Isidoro‐García M, Sanz C, García‐Solaesa V, Pascual M, Pescador DB, Lorente F, Dávila I. PTGDR gene in asthma: a functional, genetic, and epigenetic study. Allergy 2011; 66: 1553–1562.

Promoter genetic variants of prostanoid DP receptor (PTGDR) gene in patients with asthma

Background:  PTGDR gene has been identified as an asthma‐susceptibility gene. Recently, functional genetic variants have been associated with asthma. The objective of this work was to study −549T>C, −441C>T and −197T>C PTGDR promoter polymorphisms in a Spanish population.

927T>C polymorphism of the cysteinyl-leukotriene type-1 receptor (CYSLTR1) gene in children with asthma and atopic dermatitis.

Asthma and atopic dermatitis share several common features and Cysteinyl‐leukotrienes are mediators that participate in the pathogenesis of both diseases. Recently, a new polymorphism (927T>C) has been identified in cysteinyl‐leukotriene type‐1 receptor (CYSLTR1) gene. This gene is found on the X chromosome. The aim of this study was to analyze this SNP in a population of children with asthma and atopic dermatitis. In this study, 166 individuals, 79 adult controls (CTR) and 87 children with asthma (AA) were included. Forty‐one patients with asthma presented atopic dermatitis (AA‐AD). Adults were chosen as controls to confirm lack of development of asthma and allergy during childhood. Standardized history, physical examination, skin prick tests, and lung function measurements were performed in all patients. The 927T>C CYSLTR1 SNP was analyzed by direct sequencing after PCR amplification. In males (53 individuals), the C allele was significantly more common among AA‐AD patients (47%) than in CTR (8%) (Fishers p < 0.005; Monte Carlo p < 0.008; OR:9.78; 95%CI: 1.73–55.30). When comparing AA‐AD vs. AA‐NAD (patients with asthma but not atopic dermatitis), significant differences were observed, (47% vs. 15%, Fishers p = 0.014; Monte Carlo p = 0.022; OR: 4.97; 95%CI: 1.29–19.13). No differences in allele distribution were observed between these disease sub‐groups in females. The 927T>C is a silent SNP; however, it could affect transcription or translation or may be linked to an unidentified, functional polymorphism and thus may pre‐dispose male children to asthma and atopic dermatitis in our population. Further studies are needed to confirm these findings.

Dose reduction of efavirenz: an observational study describing cost–effectiveness, pharmacokinetics and pharmacogenetics

AIM Antiretroviral treatment implies a high cost to the healthcare system. The aim of this study was to evaluate the clinical and economic impact of efavirenz (EFV) dose adjustment by monitoring plasma concentrations and pharmacogenetic analysis of the 516G>T CYP2B6 polymorphism. MATERIALS & METHODS One hundred and ninety HIV patients treated with EFV were studied. Plasma EFV concentrations were measured by HPLC with ultraviolet detection, and pharmacogenetic analysis was performed by Real Time (RT)-PCR. RESULTS One hundred and ninety patients initially treated with a standard dose of EFV (600 mg/day) were studied. In 31 (16.3%) patients, EFV dose was reduced. A total of 87.1% of patients were heterozygous/homozygous carriers (GT/TT). CD4(+) count increased while the minimum steady-state plasma concentration and adverse effects decreased significantly after dose adjustment. Considering only the dose reduction, the adjustments accounted for a saving of 43,539 €/year. CONCLUSION The individualization of EFV dosage guided by genotyping 516G>T CYP2B6 and therapeutic drug monitoring could increase the efficiency of EFV use in antiretroviral treatment.

Discovery of the nonfunctional CYP2D6*31 allele in Spanish, Puerto Rican, and US Hispanic populations

BackgroundCYP2D6*31 (4042G>A, R440H) is an allelic variant of the highly polymorphic cytochrome P450 2D6 enzyme that has been associated with reduced functional activity. The US Food and Drug Administration (FDA)-cleared AmpliChip CYP450 test detects the 4042G>A single nucleotide polymorphism (SNP) but an allele assignment could not be made in two Spanish and two Puerto Rican individuals heterozygous for 4042G>A, resulting in no-calls. We aimed to resolve the CYP2D6*31 no-calls, determine the allele haplotype, and corroborate that CYP2D6*31 is associated with a poor metabolizer phenotype.MethodsCYP2D6 genotyping was carried out using the AmpliChip CYP450 test and long-range polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) platforms. Allele haplotype was determined by cloning and sequence analysis. Allele frequencies were determined in five population samples.ResultsA 6.6-kb long-range PCR product comprising the entire CYP2D6 gene and flanking regions was sequenced to determine the CYP2D6*31 haplotype. Identical sequences were obtained from both Puerto Ricans selected for sequence analysis. One Spanish individual with a CYP2D6*4/*31 genotype was phenotyped as a poor metabolizer with the CYP2D6 probe drug dextromethorphan (urinary ratio DM/DX=0.71). The frequency of CYP2D6*31 was determined in 176 Spanish (0.57%), 50 Puerto Rican (2.0%), and 150 Hispanic (0.33%) people. CYP2D6*31 was absent in 237 North American Caucasians and 154 African Americans.ConclusionsCYP2D6*31 was associated with poor metabolism of dextromethorphan in vivo, which is consistent with a previous report classifying this allelic variant as nonfunctional. The discovery of CYP2D6*31 in Spanish people only (or of Spanish ancestry) suggests that it may contribute to CYP2D6 variability in individuals of Spanish ancestry.

Interactions between genes and the environment. Epigenetics in allergy.

Epigenetics is defined as those inheritable changes occurring in gene expression, without actual modification in the genic DNA sequence. Epigenetic factors are chemically stable, potentially reversible, and can be modulated or induced by environmental factors. In the case of allergic disease, epigenetics could explain not only the discordances observed between monozygous twins but also phenomena such as incomplete penetrance, variable expression, gender and progenitor effects, and sporadic cases. In this sense, the hypothesis of hygiene is of great relevance in that it integrates genetic and epidemiological data in the context of environmental exposures. Among the different epigenetic factors, mention must be made of DNA methylation, covalent histone modifications, and other mechanisms that include different protein complexes and RNA-mediated modifications. The regulatory effect of these phenomena upon immune response has important implications for allergic diseases. At present, different lines of pharmacological research are being conducted, based on the modulation of epigenetic factors, modifying expression of the genes that encode for proteins implicated in allergic processes. Among such modulators, mention can be made of antisense oligonucleotides, ribozymes and interference RNA. The applications of epigenetics to the diagnosis and treatment of allergic disorders offer a very promising future of this specialty.

A new PTGDR promoter polymorphism in a population of children with asthma

Recently, functional genetic variants of the PTGDR gene have been associated with asthma. The objective of this work was to study polymorphisms of the promoter region of PTGDR and their haplotype and diplotype combinations in a Spanish population of children with asthma. In this study, 200 Caucasian individuals were included. Asthma was specialist–physician diagnosed according to the ATS criteria. The polymorphisms were analyzed by direct sequencing. In the study, the new polymorphism (‐613C > T) in the promoter region of PTGDR was analyzed. The CT genotype was more common in controls (17%) than in patients with asthma (1%) (p‐value = 0.0003; OR, 0.057; 95% CI, 0.007–0.441). The CCCT CCCC diplotype (promoter positions ‐613, ‐549, ‐441, and ‐197) was more frequent in the group of patients with asthma [Fisher’s p‐value = 0.012; OR, 10.24; 95% CI (1.25–83.68)]; this diplotype is unambiguous. To our knowledge, this is the first study of ‐613C > T PTGDR polymorphism in patients. This analysis provides more complete information on influence of diplotype combinations of PTGDR polymorphisms in asthma.

Analysis of 927T > C CYSLTR1 and -444A > C LTC4S polymorphisms in children with asthma

INTRODUCTION The cysteinyl leukotrienes (Cys-LTs) are potent inflammatory mediators in asthma. It has been suggested that the different response of patients to Cys-LTs inhibitors could be due to the presence of polymorphisms in the genes implicated in this pathway. METHODS In this study, polymorphisms 927T > C CYSLTR1 and -444A > C LTC4S were analysed in a Spanish population of 188 individuals (109 asthmatic children and 79 controls). Standardised history, skin prick tests and lung function measurements were performed in all patients. Genotypes were determined by sequencing after PCR amplification. RESULTS Differences were observed in 927T > C CYSLTR1, regarding the severity of asthma in males. A greater presence of allele C in the population with persistent asthma versus the control group (Fishers p-value = 0.001; Monte Carlo p-value = 0.003; OR: 12.35; 95 %CI: 2.18-70.00) was observed. Differences were also detected in the combined study of both polymorphisms, among controls and asthmatic patients (Monte Carlo p-value = 0.0002). In the group of males with asthma, an increase of AC variant (-444A LTC4S and 927C CYSLTR1) and a reduction in the AT genetic combination were detected. CONCLUSIONS The combined study of polymorphisms in genes of the leukotriene pathway could explain the differences observed in the studies reported on polymorphism -444A < C LTC4S individually analysed.