#METABOLOMICS WORKBENCH Mahamogren_20230911_215002 DATATRACK_ID:4305 STUDY_ID:ST002934 ANALYSIS_ID:AN004812 VERSION 1 CREATED_ON 10-17-2023 #PROJECT PR:PROJECT_TITLE Metabolic profiling of newborn DBS samples associated with transit and false PR:PROJECT_TITLE elevation of glutarylcarnitine (C5DC) PR:PROJECT_SUMMARY Background: Glutaric aciduria type-1 (GA-1) is a rare autosomal recessive PR:PROJECT_SUMMARY metabolic disorder caused by a glutaryl coenzyme A dehydrogenase (GCDH) PR:PROJECT_SUMMARY deficiency, affecting approximately 1 in 110,000 individuals globally. This PR:PROJECT_SUMMARY enzymatic deficiency leads to abnormal elevations of glutaryl-CoA and its PR:PROJECT_SUMMARY derivatives, specifically glutaric acid (GA), 3-hydroxyglutaric acid (3OHGA), PR:PROJECT_SUMMARY and glutarylcarnitine (C5DC). Clinical manifestations encompass macrocephaly, PR:PROJECT_SUMMARY developmental delays, and movement disorders. Early detection via genetic PR:PROJECT_SUMMARY testing and newborn screening (NBS), utilizing GA-1 biomarkers in dried blood PR:PROJECT_SUMMARY spot (DBS) samples, is vital for prompt intervention. Despite the NBS system, PR:PROJECT_SUMMARY transit-elevated C5DC-containing DBS samples from falsely suspected GA-1 PR:PROJECT_SUMMARY newborns sometimes yield normal results, posing diagnostic sensitivity and PR:PROJECT_SUMMARY specificity challenges. Consequently, there is a growing need for alternative PR:PROJECT_SUMMARY diagnostic tools. Comprehensive mass spectrometry-based untargeted metabolomics PR:PROJECT_SUMMARY offers promise in identifying additional informative biomarkers for PR:PROJECT_SUMMARY distinguishing falsely suspected GA-1 newborns from healthy counterparts. PR:PROJECT_SUMMARY Methodology: In this prospective study, we obtained DBS samples with PR:PROJECT_SUMMARY transit-elevated C5DC levels from falsely suspected GA-1 newborns (n=47) and PR:PROJECT_SUMMARY matched control DBS samples from healthy newborns (n=47) through the NBS PR:PROJECT_SUMMARY program. Metabolites were extracted and analyzed via liquid PR:PROJECT_SUMMARY chromatography-high-resolution mass spectrometry (LC-HRMS). Subsequent PR:PROJECT_SUMMARY multivariate and univariate statistical analyses and feature annotation enabled PR:PROJECT_SUMMARY biomarker and pathway investigations for significantly altered metabolites. PR:PROJECT_SUMMARY Results: Untargeted metabolomics analysis revealed alterations in 582 PR:PROJECT_SUMMARY upregulated and 546 downregulated metabolites. The commonly used GA-1 PR:PROJECT_SUMMARY biomarkers, including C5DC, exhibited no significant changes in the falsely PR:PROJECT_SUMMARY suspected GA-1 DBS samples. Conversely, 155 endogenous metabolites displayed PR:PROJECT_SUMMARY significant variations compared to the control group. Furthermore, our data PR:PROJECT_SUMMARY identified novel altered metabolic biomarkers, such as N-Palmitoylcysteine, PR:PROJECT_SUMMARY 3-hydroxylinoleoylcarnitine, Heptacarboxyporphyrin, and MG (0:0/20:1/0:0), along PR:PROJECT_SUMMARY with perturbed metabolic pathways like sphingolipid and thiamine metabolism PR:PROJECT_SUMMARY associated with the transient and falsely elevated C5DC levels in DBS samples. PR:PROJECT_SUMMARY Conclusions: Our untargeted metabolomics investigation unveiled distinct PR:PROJECT_SUMMARY metabolic pathways and biomarkers linked to the transient C5DC elevation in DBS PR:PROJECT_SUMMARY samples from falsely suspected GA-1 newborns. These findings can enhance GA-1 PR:PROJECT_SUMMARY diagnosis by serving as predictive indicators during NBS analysis. Validation PR:PROJECT_SUMMARY studies are warranted to confirm the presence of these newly identified PR:PROJECT_SUMMARY metabolic pathways and biomarkers in confirmed GA-1 cases. PR:INSTITUTE King Faisal Specialist Hospital and Research Centre (KFSHRC) PR:LAST_NAME Al Mogren PR:FIRST_NAME Maha PR:ADDRESS Zahrawi Street, Al Maather, Riyadh 11211, Saudi Arabia PR:EMAIL mahamogren@gmail.com PR:PHONE 966541205332 PR:DOI http://dx.doi.org/10.21228/M8T714 #STUDY ST:STUDY_TITLE Metabolic profiling of newborn DBS samples associated with transit and false ST:STUDY_TITLE elevation of glutarylcarnitine (C5DC) ST:STUDY_SUMMARY Background: Glutaric aciduria type-1 (GA-1) is a rare autosomal recessive ST:STUDY_SUMMARY metabolic disorder caused by a glutaryl coenzyme A dehydrogenase (GCDH) ST:STUDY_SUMMARY deficiency, affecting approximately 1 in 110,000 individuals globally. This ST:STUDY_SUMMARY enzymatic deficiency leads to abnormal elevations of glutaryl-CoA and its ST:STUDY_SUMMARY derivatives, specifically glutaric acid (GA), 3-hydroxyglutaric acid (3OHGA), ST:STUDY_SUMMARY and glutarylcarnitine (C5DC). Clinical manifestations encompass macrocephaly, ST:STUDY_SUMMARY developmental delays, and movement disorders. Early detection via genetic ST:STUDY_SUMMARY testing and newborn screening (NBS), utilizing GA-1 biomarkers in dried blood ST:STUDY_SUMMARY spot (DBS) samples, is vital for prompt intervention. Despite the NBS system, ST:STUDY_SUMMARY transit-elevated C5DC-containing DBS samples from falsely suspected GA-1 ST:STUDY_SUMMARY newborns sometimes yield normal results, posing diagnostic sensitivity and ST:STUDY_SUMMARY specificity challenges. Consequently, there is a growing need for alternative ST:STUDY_SUMMARY diagnostic tools. Comprehensive mass spectrometry-based untargeted metabolomics ST:STUDY_SUMMARY offers promise in identifying additional informative biomarkers for ST:STUDY_SUMMARY distinguishing falsely suspected GA-1 newborns from healthy counterparts. ST:STUDY_SUMMARY Methodology: In this prospective study, we obtained DBS samples with ST:STUDY_SUMMARY transit-elevated C5DC levels from falsely suspected GA-1 newborns (n=47) and ST:STUDY_SUMMARY matched control DBS samples from healthy newborns (n=47) through the NBS ST:STUDY_SUMMARY program. Metabolites were extracted and analyzed via liquid ST:STUDY_SUMMARY chromatography-high-resolution mass spectrometry (LC-HRMS). Subsequent ST:STUDY_SUMMARY multivariate and univariate statistical analyses and feature annotation enabled ST:STUDY_SUMMARY biomarker and pathway investigations for significantly altered metabolites. ST:STUDY_SUMMARY Results: Untargeted metabolomics analysis revealed alterations in 582 ST:STUDY_SUMMARY upregulated and 546 downregulated metabolites. The commonly used GA-1 ST:STUDY_SUMMARY biomarkers, including C5DC, exhibited no significant changes in the falsely ST:STUDY_SUMMARY suspected GA-1 DBS samples. Conversely, 155 endogenous metabolites displayed ST:STUDY_SUMMARY significant variations compared to the control group. Furthermore, our data ST:STUDY_SUMMARY identified novel altered metabolic biomarkers, such as N-Palmitoylcysteine, ST:STUDY_SUMMARY 3-hydroxylinoleoylcarnitine, Heptacarboxyporphyrin, and MG (0:0/20:1/0:0), along ST:STUDY_SUMMARY with perturbed metabolic pathways like sphingolipid and thiamine metabolism ST:STUDY_SUMMARY associated with the transient and falsely elevated C5DC levels in DBS samples. ST:STUDY_SUMMARY Conclusions: Our untargeted metabolomics investigation unveiled distinct ST:STUDY_SUMMARY metabolic pathways and biomarkers linked to the transient C5DC elevation in DBS ST:STUDY_SUMMARY samples from falsely suspected GA-1 newborns. These findings can enhance GA-1 ST:STUDY_SUMMARY diagnosis by serving as predictive indicators during NBS analysis. Validation ST:STUDY_SUMMARY studies are warranted to confirm the presence of these newly identified ST:STUDY_SUMMARY metabolic pathways and biomarkers in confirmed GA-1 cases. ST:INSTITUTE King Faisal Specialist Hospital and Research Centre (KFSHRC) ST:LAST_NAME Al Mogren ST:FIRST_NAME Maha ST:ADDRESS Zahrawi Street, Al Maather, Riyadh 11211, Saudi Arabia ST:EMAIL mahamogren@gmail.com ST:PHONE 966541205332 ST:SUBMIT_DATE 2023-09-11 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male and female #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 19281533_C Factor:Control RAW_FILE_NAME=19281533_C SUBJECT_SAMPLE_FACTORS - 19534501_C Factor:Control RAW_FILE_NAME=19534501_C SUBJECT_SAMPLE_FACTORS - 20419817_C Factor:Control RAW_FILE_NAME=20419817_C SUBJECT_SAMPLE_FACTORS - 20649302_C Factor:Control RAW_FILE_NAME=20649302_C SUBJECT_SAMPLE_FACTORS - 20688684_C Factor:Control RAW_FILE_NAME=20688684_C SUBJECT_SAMPLE_FACTORS - 20688815_C Factor:Control RAW_FILE_NAME=20688815_C SUBJECT_SAMPLE_FACTORS - 20771984_C Factor:Control RAW_FILE_NAME=20771984_C SUBJECT_SAMPLE_FACTORS - 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MOH00027340288_GA Factor:GA RAW_FILE_NAME=MOH00027340288_GA SUBJECT_SAMPLE_FACTORS - MOH00027354847_GA Factor:GA RAW_FILE_NAME=MOH00027354847_GA SUBJECT_SAMPLE_FACTORS - MOH00027361087_GA Factor:GA RAW_FILE_NAME=MOH00027361087_GA #COLLECTION CO:COLLECTION_SUMMARY The Institutional Review Boards at King Faisal Specialist Hospital and Research CO:COLLECTION_SUMMARY Centre (KFSHRC) in Riyadh, Saudi Arabia (RAC # 2160 027) reviewed and approved CO:COLLECTION_SUMMARY this study and its related procedures. DBS samples were obtained from the CO:COLLECTION_SUMMARY metabolomics section laboratory, Clinical Genomics Department (CGD) in the CO:COLLECTION_SUMMARY Center for Genomic Medicine (CGM) at KFSHRC as leftover material from the CO:COLLECTION_SUMMARY newborn screening program. To identify predictive metabolic profiling, newborn CO:COLLECTION_SUMMARY DBS samples found to have transit elevation of C5DC, showing normal C5DC level CO:COLLECTION_SUMMARY in the follow-up NBS testing, were used (n=47), and DBS samples from healthy CO:COLLECTION_SUMMARY controls (n=47) were included in this study for comparison purposes to predict CO:COLLECTION_SUMMARY different metabolic profiling. The two groups were age- and gender-matched, CO:COLLECTION_SUMMARY where non-newborn samples with ages more than 4 weeks or diagnosed with other CO:COLLECTION_SUMMARY inborn errors of metabolism (IMD) were excluded from this study. CO:COLLECTION_PROTOCOL_FILENAME GA_Sample Collection CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY None #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted from DBS using a standard procedure reported SP:SAMPLEPREP_SUMMARY elsewhere (14). Briefly, one punch, a size of 3.2 mm, was collected from each SP:SAMPLEPREP_SUMMARY DBS, and then metabolites were extracted by adding 250 ul (40:40:20) (methanol: SP:SAMPLEPREP_SUMMARY acetonitrile: dH2O) and mixing in a plate shaker for 2 h at RT. Meanwhile, SP:SAMPLEPREP_SUMMARY another set of punches from each sample was collected and pooled as QC samples, SP:SAMPLEPREP_SUMMARY which were prepared to control the system's stability. After that, the extracts SP:SAMPLEPREP_SUMMARY from the study and QC samples were dried using SpeedVac (Thermo Fischer, Christ, SP:SAMPLEPREP_SUMMARY Germany). The dried samples were reconstituted in 90 ul using (1:1) mobile phase SP:SAMPLEPREP_SUMMARY A: B (A: 0.1% Formic acid in dH2O, B: 0.1% Formic acid in 50% MeOH and ACN) and SP:SAMPLEPREP_SUMMARY 10 µl of internal standard mixture for metabolomics analysis. SP:SAMPLEPREP_PROTOCOL_ID GA_Metabolites extraction #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The Waters Acquity UPLC system coupled with a Xevo G2-S QTOF mass spectrometer CH:CHROMATOGRAPHY_SUMMARY equipped with an electrospray ionization source (ESI) was used to analyze the CH:CHROMATOGRAPHY_SUMMARY samples. The extracted metabolites were chromatographed using an ACQUITY UPLC CH:CHROMATOGRAPHY_SUMMARY using XSelect (100×2.1mm, 2.5 μm) column (Waters Ltd., Elstree, UK), the CH:CHROMATOGRAPHY_SUMMARY mobile phase composed of 0.1% formic acid in dH2O as solvent A, and B consists CH:CHROMATOGRAPHY_SUMMARY of 0.1% formic acid in 50% ACN: MeOH. A gradient elution schedule was run as CH:CHROMATOGRAPHY_SUMMARY follows: 0-16 min 95- 5% A, 16-19 min 5% A, 19-20 min 5-95% A, 20-22 min 95- 95% CH:CHROMATOGRAPHY_SUMMARY A, at 300 µL/min flow rate. MS spectra were acquired under positive and CH:CHROMATOGRAPHY_SUMMARY negative electrospray ionization modes (ESI+, ESI-). MS conditions were as CH:CHROMATOGRAPHY_SUMMARY follows: source temperature was 150◦C, the desolvation temperature was 500◦C CH:CHROMATOGRAPHY_SUMMARY (ESI+) or 140 (ESI−), the capillary voltage was 3.20 kV (ESI+) or 3 kV CH:CHROMATOGRAPHY_SUMMARY (ESI−), cone voltage was 40 V, desolvation gas flow was 800.0 L/h, cone gas CH:CHROMATOGRAPHY_SUMMARY flow was 50 L/h. The collision energies of low and high functions were set at CH:CHROMATOGRAPHY_SUMMARY off and 10 V to 50 V, respectively, in MSE mode. The mass spectrometer was CH:CHROMATOGRAPHY_SUMMARY calibrated with 100–1200 Da sodium formate in both ionization modes. The lock CH:CHROMATOGRAPHY_SUMMARY mass compound, leucine-enkephalin (an external reference to the ion m/z 556.2771 CH:CHROMATOGRAPHY_SUMMARY in (ESI+) and 554.2615 (ESI-)), was infused continuously, switching between the CH:CHROMATOGRAPHY_SUMMARY sample and the reference every 45 and 60 s for ESI+ and ESI-, respectively, for CH:CHROMATOGRAPHY_SUMMARY a 0.5 s scan time, a flow rate of 10 µL/min, a cone voltage of 30 V, and CH:CHROMATOGRAPHY_SUMMARY collision energy of 4 V. Data were collected in continuum mode with Masslynx™ CH:CHROMATOGRAPHY_SUMMARY V4.1 workstation (Waters Inc., Milford, Massachusetts, USA). The samples were CH:CHROMATOGRAPHY_SUMMARY acquired randomly, and a QC sample was injected after each 10 study samples to CH:CHROMATOGRAPHY_SUMMARY control the system’s variations. A mixture of several internal standards with CH:CHROMATOGRAPHY_SUMMARY a final concentration of 10 µg/ml was added to each sample, serving as a CH:CHROMATOGRAPHY_SUMMARY reference compound to help correct any variations that may occur during sample CH:CHROMATOGRAPHY_SUMMARY preparation and analysis. CH:METHODS_FILENAME GA_LCMS Metabolomics CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity UPLC XSelect HSS C18 (100 × 2.1mm, 2.5um) CH:COLUMN_TEMPERATURE 55 CH:FLOW_GRADIENT 0–16 min 95%–5% A, 16–19 min 5% A, 19–20 min 5%–95% A, and 20–22 CH:FLOW_GRADIENT min, 95%– 95% A CH:FLOW_RATE 300 μl/min. CH:SOLVENT_A 0.1% formic acid in dH2O CH:SOLVENT_B 0.1% formic acid in 50% MeOH and ACN CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Xevo-G2-S MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:MS_COMMENTS The DIA data were collected with a Masslynx™ V4.1 workstation in continuum MS:MS_COMMENTS mode (Waters Inc., Milford, MA, USA). The raw MS data were processed following a MS:MS_COMMENTS standard pipeline using the Progenesis QI v.3.0 software. MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST002934_AN004812_Results.txt UNITS:Peak Area Has m/z:Yes Has RT:Yes RT units:Minutes #END