Pooled genome-wide analysis to identify novel risk loci for pediatric allergic asthma

Background

Genome-wide association studies of pooled DNA samples were shown to be a valuable tool to identify candidate SNPs associated to a phenotype. No such study was up to now applied to childhood allergic asthma, even if the very high complexity of asthma genetics is an appropriate field to explore the potential of pooled GWAS approach.

Methodology/Principal Findings

We performed a pooled GWAS and individual genotyping in 269 children with allergic respiratory diseases comparing allergic children with and without asthma. We used a modular approach to identify the most significant loci associated with asthma by combining silhouette statistics and physical distance method with cluster-adapted thresholding. We found 97% concordance between pooled GWAS and individual genotyping, with 36 out of 37 top-scoring SNPs significant at individual genotyping level. The most significant SNP is located inside the coding sequence of C5, an already identified asthma susceptibility gene, while the other loci regulate functions that are relevant to bronchial physiopathology, as immune- or inflammation-mediated mechanisms and airway smooth muscle contraction. Integration with gene expression data showed that almost half of the putative susceptibility genes are differentially expressed in experimental asthma mouse models.

Conclusion/Significance

Combined silhouette statistics and cluster-adapted physical distance threshold analysis of pooled GWAS data is an efficient method to identify candidate SNP associated to asthma development in an allergic pediatric population.     

Impact of Protein Domains on PE_PGRS30 Polar Localization in Mycobacteria

PE_PGRS proteins are unique to the Mycobacterium tuberculosis complex and a number of other pathogenic mycobacteria. PE_PGRS30, which is required for the full virulence of M. tuberculosis (Mtb), has three main domains, i.e. an N-terminal PE domain, repetitive PGRS domain and the unique C-terminal domain. To investigate the role of these domains, we expressed a GFP-tagged PE_PGRS30 protein and a series of its functional deletion mutants in different mycobacterial species (Mtb, Mycobacterium bovis BCG and Mycobacterium smegmatis) and analysed protein localization by confocal microscopy. We show that PE_PGRS30 localizes at the mycobacterial cell poles in Mtb and M. bovis BCG but not in M. smegmatis and that the PGRS domain of the protein strongly contributes to protein cellular localization in Mtb. Immunofluorescence studies further showed that the unique C-terminal domain of PE_PGRS30 is not available on the surface, except when the PGRS domain is missing. Immunoblot demonstrated that the PGRS domain is required to maintain the protein strongly associated with the non-soluble cellular fraction. These results suggest that the repetitive GGA-GGN repeats of the PGRS domain contain specific sequences that contribute to protein cellular localization and that polar localization might be a key step in the PE_PGRS30-dependent virulence mechanism.     

An efficient and rapid procedure for plantlet regeneration from chicory mesophyll protoplasts

An efficient procedure for plantlet regeneration from chicory mesophyll protoplasts has been developed in order to perform protoplast fusion experiments. Protoplasts were isolated from a genotype of Italian red chicory (CH 363) and purified by centrifugation in a solution containing 13% (w/v) sucrose to collect uniform protoplasts in size. After 2 days culture at a density of 2×10⁴ protoplasts ml⁻¹ of liquid medium, protoplasts were cultured following three different procedures: in liquid medium, stratified in semi-solid medium, and embedded in Ca-alginate droplets. Four different media were used and culture procedures were evaluated recording the protoplast viability, protoplast division frequency and plating efficiency for each experiment. The embedding of protoplasts in Ca-alginate droplets enhanced both division frequency and plating efficiency for chicory mesophyll cells. Furthermore, this procedure shortened the cycle of plant regeneration from protoplasts, which could be completed in eight weeks. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phylogenetic relationships in Orchis and some related genera: an approach using chloroplast DNA

A chloroplast DNA restriction fragment length polymorphism analysis has been carried out on representatives species of Orchis (Orchidaceae) and of the allied genera Aceras, Dactylorhiza , and Anacamptis . One species of Cephalanthera and one of Serapias were used as outgroups. The consensus tree from a cladistic analysis showed that Orchis , as presently defined, is paraphyletic, as it contains also Aceras anthropophorum and Dactylorhiza saccifera . The genus Orchis is divided in two clades: one including O. laxiflora, O. papilionacea, O. coriophora , and O. morio in a ladderized sequence, the other showing D. saccifera at the base, followed by a clade in which a collapse of O. mascula, O. pauciflora, O. quadripunctata is sister group to a clade composed by O. italica, O. simia , and A. anthropophorum . These results, which agree to a great extent with literature evidence on chromosomes and isozymes, have been compared with various traditional systematic hypotheses for the genus.     

The genetic component of human longevity: New insights from the analysis of pathway‐based SNP‐SNP interactions

In human longevity studies, single nucleotide polymorphism (SNP) analysis identified a large number of genetic variants with small effects, yet not easily replicable in different populations. New insights may come from the combined analysis of different SNPs, especially when grouped by metabolic pathway. We applied this approach to study the joint effect on longevity of SNPs belonging to three candidate pathways, the insulin/insulin‐like growth factor signalling (IIS), DNA repair and pro/antioxidant. We analysed data from 1,058 tagging SNPs in 140 genes, collected in 1825 subjects (1,089 unrelated nonagenarians from the Danish 1905 Birth Cohort Study and 736 Danish controls aged 46–55 years) for evaluating synergic interactions by SNPsyn. Synergies were further tested by the multidimensional reduction (MDR) approach, both intra‐ and interpathways. The best combinations (FDR<0.0001) resulted those encompassing IGF1R‐rs12437963 and PTPN1‐rs6067484, TP53‐rs2078486 and ERCC2‐rs50871, TXNRD1‐rs17202060 and TP53‐rs2078486, the latter two supporting a central role of TP53 in mediating the concerted activation of the DNA repair and pro‐antioxidant pathways in human longevity. Results were consistently replicated with both approaches, as well as a significant effect on longevity was found for the GHSR gene, which also interacts with partners belonging to both IIS and DNA repair pathways (PAPPA, PTPN1, PARK7, MRE11A). The combination GHSR‐MREA11, positively associated with longevity by MDR, was further found influencing longitudinal survival in nonagenarian females (p = .026). Results here presented highlight the validity of SNP‐SNP interactions analyses for investigating the genetics of human longevity, confirming previously identified markers but also pointing to novel genes as central nodes of additional networks involved in human longevity.     

Genome-wide association study of multiple sclerosis confirms a novel locus at 5p13.1

Multiple Sclerosis (MS) is the most common progressive and disabling neurological condition affecting young adults in the world today. From a genetic point of view, MS is a complex disorder resulting from the combination of genetic and non-genetic factors. We aimed to identify previously unidentified loci conducting a new GWAS of Multiple Sclerosis (MS) in a sample of 296 MS cases and 801 controls from the Spanish population. Meta-analysis of our data in combination with previous GWAS was done. A total of 17 GWAS-significant SNPs, corresponding to three different loci were identified:HLA, IL2RA, and 5p13.1. All three have been previously reported as GWAS-significant. We confirmed our observation in 5p13.1 for rs9292777 using two additional independent Spanish samples to make a total of 4912 MS cases and 7498 controls (ORpooled = 0.84; 95%CI: 0.80-0.89; p = 1.36 × 10-9). This SNP differs from the one reported within this locus in a recent GWAS. Although it is unclear whether both signals are tapping the same genetic association, it seems clear that this locus plays an important role in the pathogenesis of MS.