A source of new diagnostic techniques for asthma in Ireland
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The objective of this thesis is to set up new diagnostic methods for the study of asthma and related respiratory conditions in an Irish clinical laboratory, and to deepen our knowledge of the pathophysiology of this illness. Asthma currently affects 300 million individuals worldwide. Asthma healthcare costs continue to spiral upwards, incurring an estimated €500 million in direct and indirect costs to the Irish economy each year. Epidemiological studies have linked the illness with vitamin D deficiency (VDD). Like VDD, it is associated with the season and outdoor sun exposure, and both conditions are highly prevalent in Ireland, a country which enjoys little sunshine. Vitamin D receptor (VDR) polymorphisms have been associated with asthma and allergy susceptibility. A deeper understanding of the pathobiology of asthma and its association with VDD might help to provide a step towards personalised medicine, and also towards the diagnosis and treatment of asthma and associated respiratory conditions. This pilot project was developed under the guidance of Dr Yury Rochev of NUIG, and it was awarded an employment-based postgraduate programme grant from the Irish Research Council. It was carried out at Biomnis Ireland. Biomnis is an independent sector clinical pathology diagnostic laboratory which uses the latest techniques and methodologies, and which is accredited by INAB to the ISO 15189 standard. Clinical supervision was provided by Dr John Faul in James Connolly Hospital. Total serum 25OHD, IgE, calcium, hsCRP, phosphate, IgA, magnesium, alkaline phosphatase and PTH levels were measured on an Abbott Architect ci8200 (ABBOTT, Abbott Park, IL, USA). ECP was analysed on the IDM Phadia 250 (Phadia AB, Uppsala, Sweden). The FBC was determined using an automated analyser Sysmex XE-2100D. DNA extraction was performed on Maxwell 16 System (Promega Corporation, Madison, WI, USA). RT-PCR testing was conducted on Applied Biosystems Real-Time PCR System 7500 Fast (Applied Biosystems, Foster City, CA, USA). Tests for IL10, IL17a, VDR and CAMP were xxvii analysed on DS2 automated ELISA (DYNEX Technologies, Chantilly, VA, USA). Patients’ samples from cross sectional studies of vitamin D status in Irish patients and healthy volunteers were used. Our team also analysed the samples from pilot double-blind, randomised, placebo-controlled trials of vitamin D supplementation in Irish adults with asthma, and in Irish asthmatic children. The Ethics Committees of the James Connolly Hospital and the National Children’s Hospital approved the studies. During the project we successfully completed verification of the total 25OHD assays on Abbott Architect. The new tests for ECP, IL10, IL17a, VDR, CAMP and for 4 SNPs were set up for diagnostic and research testing. We found associations between vitamin D levels (25OHD) and airway obstruction in adults’ asthma and BMI in healthy Irish adults. A negative association was noted between 25OHD and IgE levels in paediatric asthmatics. In general we did not observe any significant benefit of vitamin D supplementation in asthmatic children. However improvement in asthma control was noticeable in some patients with specific genotypes. We have shown an association of TaqI and ApaI polymorphisms of the VDR gene with a susceptibility to uncontrolled asthma in paediatric patients. Also, we have demonstrated that the patients with TC for TaqI, and CC and CT genotypes for ApaI, have a significantly low level of IL-10 and an increased WBC (neutrophils in particular), and that they were associated with poor asthma control. The results of our pilot study in adult asthma were in line with those for asthmatic children. Vitamin D’s role in respiratory disorders has not yet been fully investigated. Research in this area is still at an early stage, but the preliminary data seem encouraging. Further and more extensive studies, using a larger sample, will be necessary to confirm our findings, to examine links between vitamin D and VDR polymorphisms in specific asthma phenotypes, and to investigate the possibility of using VDR polymorphisms as biomarkers for susceptibility to asthma.
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