Wafathesisv6.doc - Comprehensive Metabolomics Profiling Towards Cystic Fibrosis Biomarker Discovery Abstract Cystic fibrosis(CF is a common genetic

Wafathesisv6.doc - Comprehensive Metabolomics Profiling...

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Comprehensive Metabolomics Profiling Towards Cystic Fibrosis Biomarker Discovery
Abstract Cystic fibrosis (CF) is a common genetic disease caused majorly by autosomal recessive mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. In newborn, the disease can be noticed because it appears with meconium ileus. In males, the disease causes CF diabetes mellitus (CFRD), infertility, Progressive Pulmonary disease, increased chloride, and pancreatic exocrine insufficiency. Delay and/or prevention of morbidity and/or mortality are direct and positive outcomes of newborn screening for remediable genetic conditions as it permits early identification and intervention. In the Kingdom of Saudi Arabia (KSA) and the Arabian Gulf region, cystic fibrosis (CF) is thought to be more common than previously reported. Currently, measurement of Immuno Reactive Trypsinogen (IRT) in dry blood spots (DBS) is the gold standard method for initial newborn screening for CF, followed by targeted CFTR mutation analysis. However, while IRT is a known sensitive marker for CF detection in newborns only, it is also known to be not specific to Cystic Fibrosis. As such, our study, uses high resolution untargeted mass spectrometry-based metabolomics and lipidomics profiling to identify more reliable, sensitive, and specific biomarker(s) for CF in young and adult patients with CF. The identified biomarkers will then be characterized using high-resolution tandem mass spectrometry. A prototype essay will then be developed towards the gold standard LC-MS/MS based routine newborn screening. The biomarker sensitivity, specificity, and predictability will be validated in serum and dried blood spots (DBS). This newly discovered biomarker(s) may serve as an alternative (more reliable) diagnostic biomarker with additional prognostic and therapeutic value in patients with CF. 2
Contents Abstract ....................................................................................................................................................... 2 Contents ...................................................................................................................................................... 3 List of Figures ............................................................................................................................................... 5 List of Tables ................................................................................................................................................ 6 Chapter 1: Introduction ............................................................................................................................... 7 1.1 Techniques of Cystic Fibrosis diagnosis 11 1.2 Techniques of metabolomics 13 Chapter 2: Literature Review ..................................................................................................................... 16 2.1 Biomarkers of inflammation 18 2.2 Biomarkers of infection 19 2.3 Biomarkers for the detection and prediction of exacerbations 21 2.4 Biomarkers for CF-related diabetes mellitus (CF-RDM) 21 Chapter 3: Methodology ............................................................................................................................ 23 3.1 Study Design and Patient Samples 23 3.2 Chemicals and materials: 23 3.3 Human Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) ELISA Kit 24 3.4 DELFLA Neonatal IRT Kit (Time -resolved fluoroimmunoassay): 26 3.5 Metabolomics 27 3.6 Statistical Analysis 31 Chapter 4: Results ...................................................................................................................................... 32 4.1Clinical Demographics 32 3
4.2 Human Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) ELISA: ................................... 35 4.3Neonatal IRT Screening ELISA: .............................................................................................................. 36 4.4 GC/MS metabolomics 37 4.5 LCMS/MS metabolomics 48 Chapter 5: Discussion ................................................................................................................................ 63 Chapter 6: Conclusion and Future Directions ............................................................................................ 64 References ................................................................................................................................................. 65 List of Figures 4
Figure 1 Pathophysiological cascade of respiratory disorder in cystic fibrosis (25,26) ............................................... 8 Figure 2 Flowchart of patient recruitment and study participation. PAP, pancreatitis-associated protein; PSP, pancreatic stone protein (28) ........................................................................................................................................ 12

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