Background: Asthma and allergic rhinitis (AR) are common allergic comorbidities with a strong genetic component in which epigenetic mechanisms might be involved.Objective: We aimed to identify novel risk loci for asthma and AR while accounting for parent-of-origin effect.Methods: We performed a series of genetic analyses, taking into account the parent-of-origin effect in families ascertained through asthma: (1) genome-wide linkage scan of asthma and AR in 615 European families, (2) association analysis with 1233 single nucleotide polymorphisms (SNPs) covering the significant linkage region in 162 French Epidemiological Study on the Genetics and Environment of Asthma families with replication in 154 Canadian Saguenay-Lac-Saint-Jean asthma study families, and (3) association analysis of disease and significant SNPs with DNA methylation (DNAm) at CpG sites in 40 Saguenay-Lac-Saint-Jean asthma study families.Results: We detected a significant paternal linkage of the 4q35 region to asthma and allergic rhinitis comorbidity (AAR; P = 7.2 x 10(-5)). Association analysis in this region showed strong evidence for the effect of the paternally inherited G allele of rs10009104 on AAR (P = 1.1 x 10(-5), reaching the multipletesting corrected threshold). This paternally inherited allele was also significantly associated with DNAm levels at the cg02303933 site (P = 1.7 x 10(-4)). Differential DNAm at this site was found to mediate the identified SNP-AAR association.Conclusion: By integrating genetic and epigenetic data, we identified that a differentially methylated CpG site within the melatonin receptor 1A (MTNR1A) gene mediates the effect of a paternally transmitted genetic variant on the comorbidity of asthma and AR. This study provides a novel insight into the role of epigenetic mechanisms in patients with allergic respiratory diseases.

Background: The multifactorial pathogenesis of ischaemic heart disease (IHD) includes a combination of environmental and genetic factors. Activation of inflammatory pathways is common in IHD, but mechanisms are still unknown. We and others have recently proposed that a polymorphism in the CD14 receptor gene of monocytes may be involved in IHD. In this study, we assessed whether other polymorphisms of genes codifying for proinflammatory mediators are involved in the inflammatory response observed in IHD. Methods: We studied 87 pts with Braunwald class IIIB unstable angina (Group 1) and 32 pts with stable angina (Group 2). The following polymorphisms were detected by polymerase chain reaction and restriction analysis: -174 G/C of IL6, Thr/Asn26 of TNF-~ and four polymorphisms of TNF-c~ (-376 G/A,-308 G/A,-244 G/A, -238 G/A). Genotype .and allele frequencies were correlated with C-reective protein (CRP) levels and IL-6 production by circulating monocyte in response to LPS-challenge (lng/ml for 4 hours). Results: Genotype and allele frequencies of the investigated polymorphisms were not different among groups. However, elevated levels of CRP (> 3mg/L) were observed in 61% of the overall carriers of thr/thr genotype of TNF-[5 gene and 31% of thr/asn carders (p<0.05). Moreover, IL-6 production in response to LPS was higher in GG homozygotes of IL-6 gene than in GT heterozygotes (4.4 ng/ml vs 2.0 ng/ml; p<0.05). No correlation was found between CRP levels, IL-6 production and polymorphisms of TNF-c< gene, Conclusion: Our study suggests that in a polygenic and muItifactodal syndrome such as IHD, polymorphisms of TNF-~ and IL-6 genes, but not of TNF-m are related with an enhanced pro-inflammatory response.

Background: Nasal polyposis (NP) is an inflammatory disease of the upper nasal airways frequently present in CF patients. Interferon-Related Developmental Regulator 1 (IFRD1) gene was reported as a possible modifier of CF lung disease severity. Three IFRD1 SNPs were analyzed to investigate a possible effect on the development of NP in CF patients. Methods and patients: The DNA of 143 patients with CF (40 with and 103 without NP) was purified from peripheral blood samples. IFRD1 SNPs (rs7817, rs3807213, rs6968084) were genotyped by restriction enzyme analysis. Results: The T allele of the common polymorphisms rs7817 and the rs7817-rs3807213 haplotype were associated with NP (p = 0.002 and 0.004, respectively). Conclusions: These results showed the association of the IFRD1-rs7817 polymorphism with NP in CF patients.

Several lines of evidence suggest that RBFOX1 is a key regulator of transcriptional and splicing programs in neural cells during development, and that it is expressed in a neuronal module enriched for known autism susceptibility genes. We have investigated its expression by semiquantitative RT-PCR in accessible nonbrain resources in eighteen autism spectrum disorder sib-pairs belonging to the Italian Autism Network cohort. RBFOX1 gene expression was detected in lymphoblastoid cell lines but not in lymphocytes. No significant differences between autism spectrum disorders and non-affected brothers were found. We were not able to replicate in lymphoblastoid cell lines the previously reported RBFOX1 gene downregulation in autism, even if a trend was observed. This might be due to less pronounced transcription level differences in RBFOX1 gene expression in lymphoblastoid cell lines than in brain samples.

OBJECTIVES AND DESIGN:Inflammation has a prominent role in the development of atherosclerosis. Type 2 diabetes could contribute to atherosclerosis development by promoting inflammation. This status might accelerate changes in intrinsic vascular wall cells and favor plaque formation. Cyclooxygenase 2 (COX-2) is highly expressed in atherosclerotic plaques. COX-2 gene expression is promoted through activation of toll-like receptor 4 (TLR4) and pro-inflammatory cytokine interleukin 1β (IL1-β). Aim of this study is to investigate whether expression profiles of pro-inflammatory genes such as COX-2, TLR4 and IL1-β in atherosclerotic plaques are altered in type 2 diabetes (T2D).METHODS:Total RNA was isolated from plaques of atherosclerotic patients and expression of COX-2, TLR4, IL1-β analyzed using real-time PCR. Histological analysis was performed on sections of the plaque to establish the degree of instability.RESULTS:Statistically significant differences in mRNA expression of COX-2 and IL1-β were found in plaques of T2D compared with non-T2D patients. A multi-variable linear regression model suggests that COX-2 mRNA expression is affected by T2D pathology and IL1-β mRNA expression in atherosclerotic plaques.CONCLUSIONS:Our results support the hypothesis that T2D pathology contributes in vivo to increase the inflammatory process associated with the atherosclerotic plaque formation, as shown by an increment of COX-2 and IL1-β mRNA expression.

Background. Airway inflammation is a key component of asthma that depends on the interplay of multiple genes. However, the association between health outcomes and genetic factors is usually evaluated by testing each candidate gene variant individually. Aim. To simultaneously assess the association of symptom severity with tag-SNPs located in inflammation-related genes and the FeNO level. Methods. In Verona (Italy), 327 asthmatics (aged 20-64) were identified from the general population in the GEIRD study (2008/2010). Individuals underwent a clinical interview, blood sampling for genotyping, and the measurement of FeNO (200 ml/s). SNPs in the CHI3L1, RAD50/IL4/IL5/IL13, IL33, and MS4A2 gene regions were genotyped by a custom GoldenGate Genotyping Assay. A symptom severity score (SSS) was computed by a multiple correspondence analysis on the basis of asthma-like symptoms, asthma attacks and hospitalization in the past year. The association of the SSS with SNPs and FeNO was assessed by a SEM. SNPs were summarised by a latent variable; atopy, gender, smoking habits and ICS use in the past year were considered as potential confounders. Results. The SEM is described in figure 1: Conclusions. This preliminary analysis suggests that inflammation-related genes could have a direct effect (not mediated by FeNO and atopy) on symptom severity in adult asthmatics.

Toll-like receptor-4 (TLR4) is a primary receptor of the innate immune reaction and compelling evidence demonstrates its involvement in the pathogenesis of atherosclerosis and stroke. TLR4 is constitutively expressed on monocytes and endothelial cells; it is highly expressed in atherosclerotic plaques and in peripheral blood of patients after ischemic stroke. Polymorphisms in the promoter region that alter the transcriptional regulation of this gene may represent genetic risk factors involved in the predisposition to atherosclerotic disease. In this study we investigated the effect on TLR4 gene expression of three polymorphisms in the upstream regulatory region at positions -1607T>C/rs10759932, -2026A>G/rs1927914 and -2604G>A/rs10759931 in peripheral blood of atherosclerotic patients. RNA from individuals homozygous for the -2604A allele showed a lower expression of the gene when compared to patients carrying the counterparts GG+GA. Electrophoretic mobility shift assays showed differences in the electrophoretic mobility of the DNA-nuclear protein complexes formed by the G>A variants, suggesting that the two alleles differ in their binding affinity to transcriptional factors.

BackgroundGenetic variability of the major subunit (CACNA1E) of the voltage-dependent Ca(2+) channel Ca(V)2.3 is associated to risk of type 2 diabetes, insulin resistance and impaired insulin secretion in nondiabetic subjects. The aim of the study was to test whether CACNA1E common variability affects beta cell function and/or insulin sensitivity in patients with newly diagnosed type 2 diabetes.Methodology/principal findingsIn 595 GAD-negative, drug naïve patients (mean ± SD; age: 58.5 ± 10.2 yrs; BMI: 29.9 ± 5 kg/m(2), HbA1c: 7.0±1.3) with newly diagnosed type 2 diabetes we: 1. genotyped 10 tag SNPs in CACNA1E region reportedly covering ∼93% of CACNA1E common variability: rs558994, rs679931, rs2184945, rs10797728, rs3905011, rs12071300, rs175338, rs3753737, rs2253388 and rs4652679; 2. assessed clinical phenotypes, insulin sensitivity by the euglycemic insulin clamp and beta cell function by state-of-art modelling of glucose/C-peptide curves during OGTT. Five CACNA1E tag SNPs (rs10797728, rs175338, rs2184945, rs3905011 and rs4652679) were associated with specific aspects of beta cell function (p<0.05-0.01). Both major alleles of rs2184945 and rs3905011 were each (p<0.01 and p<0.005, respectively) associated to reduced proportional control with a demonstrable additive effect (p<0.005). In contrast, only the major allele of rs2253388 was related weakly to more severe insulin resistance (p<0.05).Conclusions/significanceIn patients with newly diagnosed type 2 diabetes CACNA1E common variability is strongly associated to beta cell function. Genotyping CACNA1E might be of help to infer the beta cell functional phenotype and to select a personalized treatment.

This study aims to determine the genetic involvement in the susceptibility to asthma, rhinitis and COPD, by candidate gene association analysis, in a large and accurately defined series of Italian subjects, even considering exposure to some environmental contexts and life-styles. The study population included 1075 subjects (aged 20-66 years) from the general population, enrolled in the frame of the Gene Environment Interactions in Respiratory Diseases (GEIRD) study between 2007 and 2010. Cases and controls were diagnosed during a clinical examination that included a detailed interview, pre/post bronchodilator spirometry, methacoline challenge, skin prick tests. A panel of 384 Single Nucleotide Polymorphisms (Tag-SNP), representative of 63 candidate genes with a previous indication of possible association to the studied diseases, was genotyped by a customized GoldenGate Genotyping assay. Presently, genotyping of 725/1075 subjects are completed. A preliminary association study of candidate gene polymorphisms was conducted on these data, for the susceptibility to one or more of the studied phenotypes, by basic association test based on allele frequency comparison. Presence of association (unadjusted p

Objective The objective of this study was to replicate an association study on a newly collected Italian autism spectrum disorder (ASD) cohort by studying the genetic markers associated with ASDs from recent genome-wide and candidate gene association studies. Methods We have genotyped 746 individuals from 227 families of the Italian Autism Network using allelic discrimination TaqMan assays for seven common single-nucleotide polymorphisms: rs2292813 (SLC25A12 gene), rs35678 (ATP2B2 gene), rs4307059 (between CDH9 and CDH10 genes), rs10513025 (between SEMA5A and TAS2R1 genes), rs6872664 (PITX1 gene), rs1861972 (EN2 gene), and rs4141463 (MACROD2 gene). A family-based association study was conducted. Results A significant association was found for two of seven markers: rs4307059 T allele (odds ratio: 1.758, SE=0.236; P-value=0.017) and rs35678 TC genotype (odds ratio: 0.528, SE=0.199; P-value=0.0013). Conclusion A preferential allele transmission of two markers located at loci previously associated with social and verbal communication skill has been confirmed in patients of a new ASD family sample.

BACKGROUND:Imputation is a statistical process used to predict genotypes of loci not directly assayed in a sample of individuals. Our goal is to measure the performance of imputation in predicting the genotype of the best known gene polymorphisms involved in drug metabolism using a common SNP array genotyping platform generally exploited in genome wide association studies.METHODS:Thirty-nine (39) individuals were genotyped with both Affymetrix Genome Wide Human SNP 6.0 (AFFY) and Affymetrix DMET Plus (DMET) platforms. AFFY and DMET contain nearly 900000 and 1931 markers respectively. We used a 1000 Genomes Pilot + HapMap 3 reference panel. Imputation was performed using the computer program Impute, version 2. SNPs contained in DMET, but not imputed, were analysed studying markers around their chromosome regions. The efficacy of the imputation was measured evaluating the number of successfully imputed SNPs (SSNPs).RESULTS:The imputation predicted the genotypes of 654 SNPs not present in the AFFY array, but contained in the DMET array. Approximately 1000 SNPs were not annotated in the reference panel and therefore they could not be directly imputed. After testing three different imputed genotype calling threshold (IGCT), we observed that imputation performs at its best for IGCT value equal to 50%, with rate of SSNPs (MAF > 0.05) equal to 85%.CONCLUSIONS:Most of the genes involved in drug metabolism can be imputed with high efficacy using standard genome-wide genotyping platforms and imputing procedures.