Development of a tool for screening adverse food reactions and food allergy in Portuguese children.

INTRODUCTION AND OBJECTIVES: A standardised questionnaire may be an excellent tool for epidemiological studies aiming at screening children with suspected food allergies. Thus, the aim of the present study was to develop a screening questionnaire for assessing children with suspected food allergy and to analyse its reproducibility. MATERIALS AND METHODS: A questionnaire of adverse food reactions was developed by literary review of similar questionnaires validated in other countries as well as less well defined, non-validated Portuguese questionnaires. Peer review of the questionnaire by a panel of specialists and subsequent exploratory analysis was carried out by applying the questionnaire in children with confirmed food allergy. Test-retest analysis was performed by giving a face-to-face questionnaire to 159 children with suspected adverse food reactions, aged between three and 11 years. Temporal stability using Spearman Rho correlation test and reproducibility was studied using Cohen's Kappa index. RESULTS: 115 children confirmed adverse food reactions that occurred with one or more foods. Retest was given about three weeks after the test, to 50 of these children who were randomly selected. The questionnaire showed good temporal stability (Spearman correlation coefficient of 0.834), and good reproducibility (only two of the 27 items had a Kappa index <0.60). CONCLUSIONS: This questionnaire showed good temporal stability and reproducibility. Its validation for screening children with suspected food allergy will allow a standardised approach to diagnosis and comparison of results obtained in different centres.

Retinoic Acid Modulates PTGDR Promoter Activity.

BACKGROUND AND OBJECTIVE: Vitamin A has been linked to the development of allergic diseases although its role is not fully understood, Retinoic acid (RA), a metabolite of Vitamin A, has been previously associated with the prostaglandin pathway, and PTGDR, a receptor of PGD2, has been proposed as a candidate gene in allergy and asthma. Considering the role of PTGDR in allergy, the goal of this study was to analyze the effect of RA on the activation of the promoter region of the PTGDR gene. METHODS: A549 lung epithelial cells were transfected with 4 combinations of genetic variants of the PTGDR promoter and stimulated with all-trans RA (ATRA); luciferase assays were performed using the Dual Luciferase Reporter System, and real-time quantitative polymerase chain reaction was used to measure the expression of PTGDR, CYP26A1, RARA, RARB, RARG, and RXRA in basal A549 cell cultures and after ATRA treatment. We also performed an in silico analysis. RESULTS: After ATRA treatment increased expression of CYP26A1 (12-fold) and RARB (4-fold) was detected. ATRA activated PTGDR promoter activity in transfected cells (P<.001) and RA response element sequences were identified in silico in this promoter region. CONCLUSIONS: RA modulated PTGDR promoter activity. Differential response to RA and to new treatments based on PTGDR modulation could depend on genetic background in allergic asthmatic patients.

Analysis of FOXP3 gene in children with allergy and autoimmune diseases.

BACKGROUND: Allergy and autoimmunity are important immunological entities underlying chronic diseases in children. In some cases both entities develop simultaneously in the same patient. FOXP3 gene codes for a transcription factor involved in regulation of the immune system. Considering that regulatory T cells are involved in controlling immunological disease development, and the relevant role of FOXP3 in this kind of T cells, the objective of this study was to analyse the FOXP3 gene in the most prevalent autoimmune diseases and/or allergies in childhood in a European population. METHODS: A total of 255 Caucasian individuals, 95 controls and 160 patients diagnosed with allergic, autoimmune or both diseases were included in this study. The molecular analysis of FOXP3 was performed by DNA sequencing following the recommendations for quality of the European Molecular Genetics Quality Network. Genomic DNA was extracted from peripheral blood of all participants and was amplified using the polymerase chain reaction. After the visualisation of the amplified fragments by agarose gel-electrophoresis, they were sequenced. RESULTS: Thirteen different polymorphisms in FOXP3 gene were found, seven of which had not been previously described. The mutated allele of SNP 7340C>T was observed more frequently in the group of male children suffering from both allergic and autoimmune diseases simultaneously (p=0.004, OR=16.2 [1.34-195.15]). CONCLUSIONS: In this study we identified for first time genetic variants of FOXP3 that are significantly more frequent in children who share allergic and autoimmune diseases. These variants mainly affect regulatory sequences that could alter the expression levels of FOXP3 modifying its function including its role in Treg cells.

Polymorphisms of the IL12B, IL1B, and TNFA genes and susceptibility to asthma.

BACKGROUND: Asthma is one of the most common chronic inflammatory diseases in developed countries. Susceptibility to asthma is associated with interaction between multiple genes and environmental factors. Several cytokines play a major role in the pathophysiology of the disease. OBJECTIVE: We analyzed the distribution of cytokine gene polymorphisms in a group of patients with asthma and a control group in order to determine the effect of these variants, or their combinations, on the development of clinical phenotypes. METHODS: We genotyped 22 single-nucleotide polymorphisms (SNPs) corresponding to 13 cytokine genes (IFNG, IL1A, IL1B, IL1R1, IL1RN, IL2, IL4, IL4R, IL6, IL10, IL12B, TGFB1, and TNFA) in 376 individuals (219 asthmatic patients and 157 controls). Genetic association was evaluated using genotype and allele models for different asthma phenotypes. Gene-gene interactions were explored using multifactor dimensionality reduction. RESULTS: Genotype AC of IL12B-1188 was associated with the presence of asthma. A significant association was detected between 2 SNPs analyzed in TNFA (-308 and -238) and atopic asthma and severe-persistent asthma. The IL1B TT haplotype (3962T and -511T) was also associated with atopy and moderate-persistent asthma. CONCLUSION: Our data show that the presence of SNPs in IL12B, TNFA, and IL1B was significantly associated with asthma, atopy, and severity of asthma.We also highlight the importance of genetic context, haplotype, and gene-gene interaction analysis in genetic association studies.

Tryptase: genetic and functional considerations.

Tryptase is one of the main proteases located in the secretory granules of the mast cells, and is released through degranulation. It is therefore assumed to play an important role in inflammatory and allergic processes. Four genes are known to encode for these enzymes, with different alleles that give rise to different types of tryptases. The term "tryptase" generally refers to ß-tryptase, which in vivo is a heterotetramer, possessing a structure of vital importance for enabling drug and substrate access to the active site of the molecule. Tryptase has been reported to possess antagonistic functions, since it plays an important role both in inflammatory phenomena and as a protector against infection. In allergic processes it is associated to bronchial hyperresponsiveness in asthmatic patients, where PAR-2 is of great importance as an airway receptor. Lastly, the genes that encode for tryptase are highly polymorphic and complex. As a result, it is important to establish a relationship between genotype and phenotype in disorders such as asthma, and to identify mutations that are presumably of pharmacological relevance.

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

BACKGROUND: Asthma affects more than 300 million individuals in the world. Several studies have demonstrated the importance of the genetic component. The aim of this study is to develop a holistic approach, including genetic, epigenetic, and expression analysis to study the Prostaglandin D2 receptor gene (PTGDR) in asthmatic patients. METHODS: In this study, 637 Caucasian individuals were included. Genetic variants were characterized by sequencing, and haplotype and diplotype combinations were established. Electrophoretic mobility shift assays (EMSAs) were performed with different promoter variants. An epigenetic analysis of PTGDR was for the first time developed by MassArray assays, and gene expression was determined by real-time polymerase chain reaction. RESULTS: The -197T > C (Fisher's P = 0.028) and -613C > T (Fisher's P < 0.001) polymorphisms were found to be significantly associated with allergic asthma and allergy to pollen and mites, respectively. In addition, several haplotype and diplotype combinations were associated with different allergy and asthma phenotypes. The presence of the -613C > T SNP determined variations in the EMSAs. Moreover, consistent differences in the methylation and expression patterns were observed between asthmatic patients and controls determining a 2.34-fold increase of PTGDR gene expression in asthmatic patients. CONCLUSIONS: Genetic combinations described have functional implications in the PTGDR promoter activity by changing the transcription factors affinity that will help characterize different risk groups. The differences observed in the transcription factors affinity and in the methylation pattern bring insight into different transcription regulation in these patients. To the best of our knowledge, this is the first work in which the implication of genetic and epigenetic factors of PTGDR has been characterized pointing to putative therapeutic targets.

Relationship between airborne pollen counts and the results obtained using 2 diagnostic methods: allergen-specific immunoglobulin E concentrations and skin prick tests.

BACKGROUND: Patients with pollinosis show allergic symptoms related to airborne pollen levels, although this association is not always close. The use of new diagnostic techniques could improve our knowledge of this relationship. OBJECTIVE: To evaluate the relationship between pollen counts and the results obtained using 2 diagnostic techniques: the skin prick test and allergen-specific immunoglobulin E (slgE) concentrations in serum. METHODS: Sixty-eight pollen-allergic patients were diagnosed using a combination of the high-capacity screening approach ADVIA Centaur with a panel of 13 purified allergens and a skin prick test (SPT) with conventional extracts. Pollen levels were obtained by means of a volumetric sampler. RESULTS: The highest percentages of sensitization were detected for grass mixture allergens and major recombinant grass allergens (Phl p 1 and Phi p 5), followed by olive tree extracts and olive allergens (Ole e 1 and Ole e 9), in SPT and using recombinant allergens, respectively. The main pollen types registered in the atmosphere during 2006 and 2007 were Quercus, Poaceae, and Cupressaceae. A statistically significant correlation was observed between total pollen levels and median values of slgE, especially in 2007. CONCLUSION: A strong and significant positive correlation was found between pollen counts and slgE levels. This correlation was weaker in the case of SPT and airborne pollen.

Usefulness of endoscopic ultrasonography (EUS) for selecting carcinoid tumors as candidates to endoscopic resection.

INTRODUCTION: Carcinoid tumors (CTs) represent the most common type of neuroendocrine tumors (NETs). Digestive CTs in the gastroduodenal and colorectal tracts may be assessed using endoscopy and echoendoscopy or endoscopic ultrasonography (EUS) with the goal of attempting local resection with curative intent without having recourse to surgery. OBJECTIVE: Endpoints in this study included:--Assessing the usefulness of EUS for selecting CTs as candidates to endoscopic excision. --Assessing the effectiveness of local resection (complete carcinoid resection) and the safety (complications) of the technique involved. PATIENTS AND METHODS: OUr series included 18 patients (12 males and 6 females) with 23 tumors. Sixteen patients (10 males and 6 females) were selected, with age ranging from 40 to 81 years (mean: 57 years), biopsied, endoscopically treated digestive carcinoid tumors, and a previous negative extension study. Twenty-one 2-to-20-mm (mean size 8 mm) tumors were resected in 23 procedures. After endoscopy plus biopsy and echoendoscopy (EUS), excision was carried out with conventional polypectomy snare mucosectomy and submucosal injection with saline and/or adrenaline in most cases (15), and mucosectomy technique following lesion ligation with elastic bands for six cases. Two cases underwent transanal endoscopic surgery (TEM), one of them following non-curative polypectomy. A total of 23 local procedures were performed with the key goal of assessing efficacy (complete resection: CR) and safety (complications). RESULTS: There were no severe complications except for the last gastric mucosectomy for a 6-mm carcinoid, where a miniperforation occurred that was solved by using 3 clips (1/23: 4.3%).EUS sensitivity was 94%. Complete resection was 90.5% (19/21). CONCLUSIONS: The endoscopic mucosal resection of selected carcinoid tumors is a safe, effective technique. EUS is the technique of choice to select patients eligible for endoscopic resection (carcinoids smaller than 20 mm in superficial layers, with an unscathed muscularis propria and negative extension study).

Genetic analysis of BRCA1 and BRCA2 in breast/ovarian cancer families from Aragon (Spain): two novel truncating mutations and a large genomic deletion in BRCA1.

We screened BRCA1 and BRCA2 germline mutations in 60 high-risk breast and/or ovarian cancer patients and 20 relatives from Aragon (Spain) by DHPLC (Denaturing High Performance Liquid Chromatography) and direct sequencing of the entire coding sequence and the splicing sites of both genes. We have identified 17 different pathogenic mutations: 8 in BRCA1 and 9 in BRCA2 in 60 unrelated patients and 50% of relatives were carriers. The prevalence of pathogenic mutations in this study was 33.33%. Two truncating mutations are novel: c.5024_5025delGA in exon 16 of BRCA1 and c.2929delC in exon 11 of BRCA2 (numbered after GenBank U14680 and U43746). Multiplex Ligation Dependent Probe Amplification (MLPA) was performed for large mutational scanning of both genes and a large genomic deletion in BRCA1 was found (DelEx8-13). Furthermore, five mutations are described for the first time in Spanish population: c.1191delC, c.3478_3479delTT and c.6633_6637delCTTAA (BRCA1) and c.3972_3975delTGAG and 3908_3909delTG (BRCA2). Three mutations have been reported previously once in Spain: c.3600_3610del11 (BRCA1), c.5804_5807delTTAA (BRCA2) and c.9246C>A (BRCA2). The mutation c.5374_5377delTATG has been found before only in two unrelated families from Castilla-Leon, Spain (BRCA2). Frequent mutations described in Spanish population have also been present: c.187_188delAG, c.5242C>A and c.5385insC in BRCA1 and c.3492_3493insT and c.9254_9258delATCAT in BRCA2. c.5242C>A, 3972_3975delTGAG and c.5804_5807delTTAA were the recurrent mutations found. Fifteen different unclassified variants were identified (25% families). Although specific BRCA1 and BRCA2 mutations are recurrently reported as a result of genetic founder effects we conclude that heterogeneous ethnicity increases the variety of mutations that can be found in Spanish populations.