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Metabolomics for discovery of biomarkers for cystic fibrosis: Towards MS-based primary screening methods with improved positive predictive value
Abstracts
Metabolomics for discovery of biomarkers for cystic fibrosis: Towards MS-based primary screening methods with improved positive predictive value Alicia DiBattista a, Adriana N. Macedo a, Osama Y. Al-Dirbashi b, Pranesh Chakraborty b, Philip Britz-McKibbin a,* a Department of Chemistry and Chemical Biology, McMaster University, Hamilton, ON, Canada b Department of Pediatrics, Children's Hospital of Eastern Ontario, Ottawa, ON, Canada Objectives: Due to benefits derived from early therapeutic intervention, many newborn screening programs have recently included cystic fibrosis (CF) within an expanded panel of genetic diseases for population screening. Primary screening approaches currently adopt a two-tiered methodology comprised of an immunoreactive trypsin immunoassay in conjunction with a gene mutation screen. However, these methods suffer from high rates of false-positives with only 10% of screen-positive patients exhibiting elevated sweat chloride results during confirmatory diagnosis. Herein, untargeted metabolomic profiling (i.e., metabolomics) was performed on dried blood spot extracts derived from CF patients, as well as healthy/normal and IEM controls. Putative biomarkers associated with CF were identified and validated in order to allow for multiplexed screening by electrospray ionization-mass spectrometry (ESI-MS). Methods: Multi-segmented injection-capillary electrophoresismass spectrometry (MSI-CE-MS) was used as high-throughput platform for biomarker discovery from methanol extracts of DBS specimens. Multivariate statistical methods and receiver operating characteristic curves were applied for ranking single or ratiometric biomarkers in terms of their sensitivity and specificity for CF classification. Results: Several polar metabolites were found to be differentially expressed in DBS extracts of CF patients relative to healthy controls and several IEM controls. These metabolites serve as useful diagnostic markers of CF while also providing deeper insight into the underlying mechanisms associated with CFTR gene expression and disease progression. Conclusions: This work demonstrates that CF is associated with specific perturbations in metabolism that offers a costeffective strategy for primary screening with improved positive predictive value when using MS infrastructure at newborn screening facilities.
doi:10.1016/j.clinbiochem.2014.07.055
Metabolomics for improved patient stratification in cystic fibrosis: Characterization of the sweat metabolome Adriana N. Macedo*, Alicia DiBattista, Karen P. Lam, Stephen Hill, Linda Pedder, Philip Britz-McKibbin McMaster University, Hamilton, Ontario, Canada Objectives: The sweat chloride test remains the gold standard in diagnosis of cystic fibrosis (CF) in screen-positive infants in expanded newborn screening programs. However, it is susceptible to ambiguous results when chloride is present at intermediate levels (30–59 mM), which occurs frequently in cases of mild onsets (pancreatic sufficient CF and CF-related disease) or heterozygous patients with no manifestation of CF. Therefore, the aim of this study was to identify and accurately quantify metabolites present in sweat, which are differentially expressed in CF patients, healthy controls and patients with mild onsets, using an untargeted metabolic profiling approach.
143
Methods: Pilocarpine iontophoresis sweat samples collected at the McMaster Children's Hospital were analyzed using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). This unique approach allows the serial injection of seven sample plugs in a single run, offering advantages in terms of sample throughput and data quality, as several samples can be analyzed within a single separation in addition to blanks or pooled samples for quality control. Results: A comparison of sweat samples with low, intermediate and high concentration of chloride has shown metabolites (mainly amino acids) that are characteristically up or down regulated in CF patients relative to controls (low chloride), which can be used to differentiate these two groups from the intermediate chloride. Conclusions: Besides chloride, there are several other metabolites present in sweat, which can serve as putative markers to enhance CF diagnosis and patient stratification, which is critical for informing treatment decisions notably for patients with ambiguous intermediate chloride results. doi:10.1016/j.clinbiochem.2014.07.056
Rapid confirmatory testing of thyroxine status by multi-segment capillary electrophoresis-mass spectrometry with chemical derivatization Meera Shanmuganathan a,*, Philip Britz-McKibbin a, Osama Y. Al-Dirbashi b, c a McMaster University, Hamilton, Ontario, Canada b Children's Hospital of Eastern Ontario, Ontario, Ottawa, Canada c University of Ottawa, Ottawa, Ontario, Canada Objectives: Currently in North America, confirmatory testing for congenital hypothyroidism (CH) relies on fluorescence-based immunoassays for total thyroxine (T4) hormone determination that is limited by cross-reactivity due to matrix interferences. Routine analysis by electrospray tandem mass spectrometry (ESIMS/MS) is challenging since T4 is acid-labile and heat sensitive precluding conventional acid-catalyzed butylation without degradation. Herein, we introduce a high-throughput approach for accurate quantification of T4 in dried blood spot extracts (DBS) using multi-segment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) couple with a novel chemical derivatization strategy. Methods: In order to reliably detect low nanomolar levels of total T4, a rapid chemical derivatization method that occurs under ambient chemical conditions was developed as a way to enhance ionization efficiency of labeled-T4 under positive ion mode. Also, separations are performed under alkaline conditions in order to ensure stability of T4 without degradation. MSI-CE-MS method was used to simultaneous analyze multiple dried blood spot extracts including a healthy control as reference with an effective throughput of 3 min/sample. Results: Rigorous optimization of reaction, extraction and ionization conditions was examined while ensuring maximum recovery of T4 without bias. The performance of MSI-CE-MS to measure normal and deficient thyroid hormone status in randomly assigned DBS specimens was also evaluated with rigorous clinical validation of method performance. Conclusions: A sensitive, specific and high-throughput method was developed for confirmatory testing of low nanomolar levels of T4 in DBS extracts when using chemical derivatization in conjunction with multiplexed separations based on MSI-CE-MS. doi:10.1016/j.clinbiochem.2014.07.057
Metabolomics for discovery of biomarkers for cystic fibrosis: Towards MS-based primary screening methods with improved positive predictive value Alicia DiBattista a, Adriana N. Macedo a, Osama Y. Al-Dirbashi b, Pranesh Chakraborty b, Philip Britz-McKibbin a,* a Department of Chemistry and Chemical Biology, McMaster University, Hamilton, ON, Canada b Department of Pediatrics, Children's Hospital of Eastern Ontario, Ottawa, ON, Canada Objectives: Due to benefits derived from early therapeutic intervention, many newborn screening programs have recently included cystic fibrosis (CF) within an expanded panel of genetic diseases for population screening. Primary screening approaches currently adopt a two-tiered methodology comprised of an immunoreactive trypsin immunoassay in conjunction with a gene mutation screen. However, these methods suffer from high rates of false-positives with only 10% of screen-positive patients exhibiting elevated sweat chloride results during confirmatory diagnosis. Herein, untargeted metabolomic profiling (i.e., metabolomics) was performed on dried blood spot extracts derived from CF patients, as well as healthy/normal and IEM controls. Putative biomarkers associated with CF were identified and validated in order to allow for multiplexed screening by electrospray ionization-mass spectrometry (ESI-MS). Methods: Multi-segmented injection-capillary electrophoresismass spectrometry (MSI-CE-MS) was used as high-throughput platform for biomarker discovery from methanol extracts of DBS specimens. Multivariate statistical methods and receiver operating characteristic curves were applied for ranking single or ratiometric biomarkers in terms of their sensitivity and specificity for CF classification. Results: Several polar metabolites were found to be differentially expressed in DBS extracts of CF patients relative to healthy controls and several IEM controls. These metabolites serve as useful diagnostic markers of CF while also providing deeper insight into the underlying mechanisms associated with CFTR gene expression and disease progression. Conclusions: This work demonstrates that CF is associated with specific perturbations in metabolism that offers a costeffective strategy for primary screening with improved positive predictive value when using MS infrastructure at newborn screening facilities.
doi:10.1016/j.clinbiochem.2014.07.055
Metabolomics for improved patient stratification in cystic fibrosis: Characterization of the sweat metabolome Adriana N. Macedo*, Alicia DiBattista, Karen P. Lam, Stephen Hill, Linda Pedder, Philip Britz-McKibbin McMaster University, Hamilton, Ontario, Canada Objectives: The sweat chloride test remains the gold standard in diagnosis of cystic fibrosis (CF) in screen-positive infants in expanded newborn screening programs. However, it is susceptible to ambiguous results when chloride is present at intermediate levels (30–59 mM), which occurs frequently in cases of mild onsets (pancreatic sufficient CF and CF-related disease) or heterozygous patients with no manifestation of CF. Therefore, the aim of this study was to identify and accurately quantify metabolites present in sweat, which are differentially expressed in CF patients, healthy controls and patients with mild onsets, using an untargeted metabolic profiling approach.
143
Methods: Pilocarpine iontophoresis sweat samples collected at the McMaster Children's Hospital were analyzed using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). This unique approach allows the serial injection of seven sample plugs in a single run, offering advantages in terms of sample throughput and data quality, as several samples can be analyzed within a single separation in addition to blanks or pooled samples for quality control. Results: A comparison of sweat samples with low, intermediate and high concentration of chloride has shown metabolites (mainly amino acids) that are characteristically up or down regulated in CF patients relative to controls (low chloride), which can be used to differentiate these two groups from the intermediate chloride. Conclusions: Besides chloride, there are several other metabolites present in sweat, which can serve as putative markers to enhance CF diagnosis and patient stratification, which is critical for informing treatment decisions notably for patients with ambiguous intermediate chloride results. doi:10.1016/j.clinbiochem.2014.07.056
Rapid confirmatory testing of thyroxine status by multi-segment capillary electrophoresis-mass spectrometry with chemical derivatization Meera Shanmuganathan a,*, Philip Britz-McKibbin a, Osama Y. Al-Dirbashi b, c a McMaster University, Hamilton, Ontario, Canada b Children's Hospital of Eastern Ontario, Ontario, Ottawa, Canada c University of Ottawa, Ottawa, Ontario, Canada Objectives: Currently in North America, confirmatory testing for congenital hypothyroidism (CH) relies on fluorescence-based immunoassays for total thyroxine (T4) hormone determination that is limited by cross-reactivity due to matrix interferences. Routine analysis by electrospray tandem mass spectrometry (ESIMS/MS) is challenging since T4 is acid-labile and heat sensitive precluding conventional acid-catalyzed butylation without degradation. Herein, we introduce a high-throughput approach for accurate quantification of T4 in dried blood spot extracts (DBS) using multi-segment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) couple with a novel chemical derivatization strategy. Methods: In order to reliably detect low nanomolar levels of total T4, a rapid chemical derivatization method that occurs under ambient chemical conditions was developed as a way to enhance ionization efficiency of labeled-T4 under positive ion mode. Also, separations are performed under alkaline conditions in order to ensure stability of T4 without degradation. MSI-CE-MS method was used to simultaneous analyze multiple dried blood spot extracts including a healthy control as reference with an effective throughput of 3 min/sample. Results: Rigorous optimization of reaction, extraction and ionization conditions was examined while ensuring maximum recovery of T4 without bias. The performance of MSI-CE-MS to measure normal and deficient thyroid hormone status in randomly assigned DBS specimens was also evaluated with rigorous clinical validation of method performance. Conclusions: A sensitive, specific and high-throughput method was developed for confirmatory testing of low nanomolar levels of T4 in DBS extracts when using chemical derivatization in conjunction with multiplexed separations based on MSI-CE-MS. doi:10.1016/j.clinbiochem.2014.07.057