Novel Quantitative Real-Time LCR for the Sensitive Detection of SNP Frequencies in Pooled DNA: Method Development, Evaluation and Application

BACKGROUND: Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. METHODS: The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. CONCLUSIONS: The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. SIGNIFICANCE: The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food

Prenatal exposures and exposomics of asthma

This review examines the causal investigation of preclinical development of childhood asthma using exposomic tools. We examine the current state of knowledge regarding early-life exposure to non-biogenic indoor air pollution and the developmental modulation of the immune system. We examine how metabolomics technologies could aid not only in the biomarker identification of a particular asthma phenotype, but also the mechanisms underlying the immunopathologic process. Within such a framework, we propose alternate components of exposomic investigation of asthma in which, the exposome represents a reiterative investigative process of targeted biomarker identification, validation through computational systems biology and physical sampling of environmental medi

Metabolomics applied to urine samples in childhood asthma; differentiation between asthma phenotypes and identification of relevant metabolites.

Asthma is a heterogeneous disorder and one of the most common chronic childhood diseases. An improved characterization of asthma phenotypes would be invaluable for the understanding of the pathogenic mechanisms and the correct treatment of this disease. The aim of this pilot study was to explore the potential of metabolomics applied to urine samples in characterizing asthma, and to identify the most representative metabolites. Urine samples of 41 atopic asthmatic children (further subdivided in sub-groups according to the symptoms) and 12 age-matched controls were analyzed. Untargeted metabolic profiles were collected by LC-MS, and studied by multivariate analysis. The group of the asthmatics was differentiated by a model that proved to be uncorrelated with the chronic assumption of controller drugs on the part of the patients. The distinct sub-groups were also appropriately modeled. Further investigations revealed a reduced excretion of urocanic acid, methyl-imidazoleacetic acid and a metabolite resembling the structure of an Ile\u2013Pro fragment in the asthmatics. The meaning of these findings was discussed and mainly correlated with the modulation of immunity in asthma. Metabolic profiles from urines have revealed the potential to characterize asthma and enabled the identification of metabolites that may have a role in the underlying inflammation

Inside Jacob’s story: exploring counsellor contribution to narrative co-construction using imaginary dialogues with a Biblical character!

Psychotherapeutic practice often involves the telling and retelling of a client’s stories of life in collaborative, meaning-making dialogue with a counsellor. This study demonstrates and explores the dynamics of counsellor contribution to this narrative co-construction, particularly the ways in which the counsellor’s inner conversations, reflexivity and interpretive style may emerge in practice and have an influence on the client’s understanding, re-evaluation and cohering of his or her own story. The multi-voiced, multi-layered intersubjective space and time in which this kind of narrative collaboration takes place is a difficult area to access for study but one whose potential impact on the client should make it the focus of respectful, ethical monitoring and careful reflective practice. Using phenomenological theories of reader-response and dialogical play, my research sets up an analogy between the way a reader might reflexively interact with life story episodes in a written text and the ways a counsellor might listen to and interpret a client’s stories of life over the course of a counselling contract. My project uses a comprehensive and episode-rich story of a life, the iconic ‘womb to tomb’ story of Jacob in the book of Genesis. My own hearer/reader response to the story gives rise to the creation of a set of imaginary dialogues between two interlocutors, Jacob as an elderly client reviewing his life story and myself as counsellor, listening to his stories of life. This methodology is used as a means to access an in vivo lived experience, as it might unfold in practice, of my counsellor contribution to Jacob’s story and the interplay of voices and standpoints which characterise it. Attention is drawn to the inchoate, but deeply human, intersubjective aspects of narrative co-construction as a process and the value of this form of reflective practice to surface actual praxis experience for analysis. Insights surfaced by this reader-response methodology point to the significant extent to which the hermeneutical standpoints and dialogical voices of a counsellor are actively involved and implicated in narrative co-construction

Two patients with COMT inhibitor\u2013induced hepatic dysfunction and UGT1A9 genetic polymorphism

The authors report two cases of catechol-O-methyltransferase (COMT) inhibitor-induced asymptomatic hepatic dysfunction in women with Parkinson disease. The patients were genotyped for the UDP-glucuronosyltransferase (UGT) 1A9 gene (which encodes the main COMT inhibitor-metabolizing enzyme), and found to carry mutations leading to defective glucuronidation activity. This suggests that UGT1A9 poor metabolizer genotype(s) may be a predisposing factor for COMT inhibitor-induced hepatotoxicit