#METABOLOMICS WORKBENCH ReemAlMalki91_20230411_093506 DATATRACK_ID:3857 STUDY_ID:ST002557 ANALYSIS_ID:AN004213 PROJECT_ID:PR001649 VERSION 1 CREATED_ON April 11, 2023, 10:15 am #PROJECT PR:PROJECT_TITLE Untargeted Metabolomics Identifies Biomarkers for MCADD Neonates in Dried Blood PR:PROJECT_TITLE Spots PR:PROJECT_TYPE newborn screening PR:PROJECT_SUMMARY Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common PR:PROJECT_SUMMARY inherited mitochondrial metabolic disease of fatty acid β-oxidation, especially PR:PROJECT_SUMMARY in newborns. MCADD is clinically diagnosed using Newborn Bloodspot Screening PR:PROJECT_SUMMARY (NBS) and genetic testing. Still, these methods have limitations, such as false PR:PROJECT_SUMMARY negatives or positives in NBS and variants of uncertain significance in genetic PR:PROJECT_SUMMARY testing. Thus, complementary diagnostic approaches for MCADD are needed. PR:PROJECT_SUMMARY Recently, untargeted metabolomics has been proposed as a diagnostic approach for PR:PROJECT_SUMMARY inherited metabolic diseases (IMDs) due to its ability to detect a wide range of PR:PROJECT_SUMMARY metabolic alterations. We performed untargeted metabolic profiling of dried PR:PROJECT_SUMMARY blood spots (DBS) from MCADD newborns (n=14) and healthy controls (n=14) to PR:PROJECT_SUMMARY discover potential metabolic biomarkers/pathways associated with MCADD. PR:PROJECT_SUMMARY Extracted metabolites from DBS samples were analyzed using UPLC-QToF-MS for PR:PROJECT_SUMMARY untargeted metabolomics analyses. Multivariate and univariate analyses were used PR:PROJECT_SUMMARY to analyze the metabolomics data, and pathway and biomarker analyses were also PR:PROJECT_SUMMARY performed on the significantly endogenous identified metabolites. MCADD newborns PR:PROJECT_SUMMARY had 1034 significantly dysregulated metabolites compared to healthy newborns PR:PROJECT_SUMMARY (Moderated t-test, no correction, p-value ≤ 0.05, FC 1.5). 23 endogenous PR:PROJECT_SUMMARY metabolites were upregulated, while 84 endogenous metabolites were PR:PROJECT_SUMMARY downregulated. Pathway analyses showed phenylalanine, tyrosine, and tryptophan PR:PROJECT_SUMMARY biosynthesis as the most affected pathway. Potential metabolic biomarkers for PR:PROJECT_SUMMARY MCADD were PGP (a21:0/PG/F1alpha) and glutathione with an area under the curve PR:PROJECT_SUMMARY (AUC) of 0.949 and 0.898, respectively. PGP (a21:0/PG/F1alpha) was the only PR:PROJECT_SUMMARY oxidized lipid in the top-15 biomarker list with the highest p-value and FC. PR:PROJECT_SUMMARY Also, glutathione was chosen to indicate oxidative stress events that could PR:PROJECT_SUMMARY happen during fatty acid oxidation defects. Our findings suggest that MCADD PR:PROJECT_SUMMARY newborns may have oxidative stress events as signs of the disease. However, PR:PROJECT_SUMMARY further validations of these biomarkers are needed in future studies to ensure PR:PROJECT_SUMMARY their accuracy and reliability as complementary markers with established MCADD PR:PROJECT_SUMMARY markers for clinical diagnosis. PR:INSTITUTE King Faisal Specialist Hospital and Research Centre (KFSHRC) PR:LAST_NAME AlMalki PR:FIRST_NAME Reem PR:ADDRESS King Fahad road, Riyadh 11211, Saudi Arabia PR:EMAIL 439203044@student.ksu.edu.sa PR:PHONE +966534045397 #STUDY ST:STUDY_TITLE Untargeted Metabolomics Identifies Biomarkers for MCADD Neonates in Dried Blood ST:STUDY_TITLE Spots ST:STUDY_TYPE Newborn screening ST:STUDY_SUMMARY Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common ST:STUDY_SUMMARY inherited mitochondrial metabolic disease of fatty acid β-oxidation, especially ST:STUDY_SUMMARY in newborns. MCADD is clinically diagnosed using Newborn Bloodspot Screening ST:STUDY_SUMMARY (NBS) and genetic testing. Still, these methods have limitations, such as false ST:STUDY_SUMMARY negatives or positives in NBS and variants of uncertain significance in genetic ST:STUDY_SUMMARY testing. Thus, complementary diagnostic approaches for MCADD are needed. ST:STUDY_SUMMARY Recently, untargeted metabolomics has been proposed as a diagnostic approach for ST:STUDY_SUMMARY inherited metabolic diseases (IMDs) due to its ability to detect a wide range of ST:STUDY_SUMMARY metabolic alterations. We performed untargeted metabolic profiling of dried ST:STUDY_SUMMARY blood spots (DBS) from MCADD newborns (n=14) and healthy controls (n=14) to ST:STUDY_SUMMARY discover potential metabolic biomarkers/pathways associated with MCADD. ST:STUDY_SUMMARY Extracted metabolites from DBS samples were analyzed using UPLC-QToF-MS for ST:STUDY_SUMMARY untargeted metabolomics analyses. Multivariate and univariate analyses were used ST:STUDY_SUMMARY to analyze the metabolomics data, and pathway and biomarker analyses were also ST:STUDY_SUMMARY performed on the significantly endogenous identified metabolites. MCADD newborns ST:STUDY_SUMMARY had 1034 significantly dysregulated metabolites compared to healthy newborns ST:STUDY_SUMMARY (Moderated t-test, no correction, p-value ≤ 0.05, FC 1.5). 23 endogenous ST:STUDY_SUMMARY metabolites were upregulated, while 84 endogenous metabolites were ST:STUDY_SUMMARY downregulated. Pathway analyses showed phenylalanine, tyrosine, and tryptophan ST:STUDY_SUMMARY biosynthesis as the most affected pathway. Potential metabolic biomarkers for ST:STUDY_SUMMARY MCADD were PGP (a21:0/PG/F1alpha) and glutathione with an area under the curve ST:STUDY_SUMMARY (AUC) of 0.949 and 0.898, respectively. PGP (a21:0/PG/F1alpha) was the only ST:STUDY_SUMMARY oxidized lipid in the top-15 biomarker list with the highest p-value and FC. ST:STUDY_SUMMARY Also, glutathione was chosen to indicate oxidative stress events that could ST:STUDY_SUMMARY happen during fatty acid oxidation defects. Our findings suggest that MCADD ST:STUDY_SUMMARY newborns may have oxidative stress events as signs of the disease. However, ST:STUDY_SUMMARY further validations of these biomarkers are needed in future studies to ensure ST:STUDY_SUMMARY their accuracy and reliability as complementary markers with established MCADD ST:STUDY_SUMMARY markers for clinical diagnosis. ST:INSTITUTE King Faisal Specialist Hospital and Research Centre (KFSHRC) ST:LAST_NAME AlMalki ST:FIRST_NAME Reem ST:ADDRESS Zahrawi Street, Al Maather, Riyadh 11211, Saudi Arabia ST:EMAIL 439203044@student.ksu.edu.sa ST:PHONE 0534045397 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 28 #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]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - DR_ Rajaa_MCAD_21241295 Genotype:MCADD RAW_FILE_NAME=DR_ Rajaa_MCAD_21241295 SUBJECT_SAMPLE_FACTORS - DR_Rajaa _MCAD_19505374 Genotype:MCADD RAW_FILE_NAME=DR_Rajaa _MCAD_19505374 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_ MCAD 21241125 Genotype:MCADD RAW_FILE_NAME=DR_Rajaa_ MCAD 21241125 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_ MCAD_20325183 Genotype:MCADD RAW_FILE_NAME=DR_Rajaa_ MCAD_20325183 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_ MCAD_20736112 Genotype:MCADD RAW_FILE_NAME=DR_Rajaa_ MCAD_20736112 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_ MCAD_21805686 Genotype:MCADD RAW_FILE_NAME=DR_Rajaa_ MCAD_21805686 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_MCAD_19031293 Genotype:MCADD RAW_FILE_NAME=DR_Rajaa_MCAD_19031293 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_MCAD_20296632 Genotype:MCADD RAW_FILE_NAME=DR_Rajaa_MCAD_20296632 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_MCAD_20400509 Genotype:MCADD RAW_FILE_NAME=DR_Rajaa_MCAD_20400509 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_MCAD_20725912 Genotype:MCADD RAW_FILE_NAME=DR_Rajaa_MCAD_20725912 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_MCAD_21112823 Genotype:MCADD RAW_FILE_NAME=DR_Rajaa_MCAD_21112823 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_MCAD_21245015 Genotype:MCADD RAW_FILE_NAME=DR_Rajaa_MCAD_21245015 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_MCAD_21905959 Genotype:MCADD RAW_FILE_NAME=DR_Rajaa_MCAD_21905959 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_MCAD_183489111 Genotype:MCADD RAW_FILE_NAME=DR_Rajaa_MCAD_183489111 SUBJECT_SAMPLE_FACTORS - DR_ Rajaa_ MCAD_21741306 Genotype:Ctrl RAW_FILE_NAME=DR_ Rajaa_ MCAD_21741306 SUBJECT_SAMPLE_FACTORS - DR_ Rajaa_ MCAD_21753806 Genotype:Ctrl RAW_FILE_NAME=DR_ Rajaa_ MCAD_21753806 SUBJECT_SAMPLE_FACTORS - DR_ Rajaa_MCAD_20802600 Genotype:Ctrl RAW_FILE_NAME=DR_ Rajaa_MCAD_20802600 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_ MCAD_20845942 Genotype:Ctrl RAW_FILE_NAME=DR_Rajaa_ MCAD_20845942 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_ MCAD_21747425 Genotype:Ctrl RAW_FILE_NAME=DR_Rajaa_ MCAD_21747425 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_MCAD_19031569 Genotype:Ctrl RAW_FILE_NAME=DR_Rajaa_MCAD_19031569 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_MCAD_20427551 Genotype:Ctrl RAW_FILE_NAME=DR_Rajaa_MCAD_20427551 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_MCAD_20845951 Genotype:Ctrl RAW_FILE_NAME=DR_Rajaa_MCAD_20845951 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_MCAD_21027862 Genotype:Ctrl RAW_FILE_NAME=DR_Rajaa_MCAD_21027862 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_MCAD_21379950 Genotype:Ctrl RAW_FILE_NAME=DR_Rajaa_MCAD_21379950 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_MCAD_21730638 Genotype:Ctrl RAW_FILE_NAME=DR_Rajaa_MCAD_21730638 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_MCAD_21753499 Genotype:Ctrl RAW_FILE_NAME=DR_Rajaa_MCAD_21753499 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_MCAD_21766545 Genotype:Ctrl RAW_FILE_NAME=DR_Rajaa_MCAD_21766545 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_MCAD_21780952 Genotype:Ctrl RAW_FILE_NAME=DR_Rajaa_MCAD_21780952 SUBJECT_SAMPLE_FACTORS - - Genotype:- RAW_FILE_NAME=- #COLLECTION CO:COLLECTION_SUMMARY DBS samples were obtained from the metabolomics section in the Center for CO:COLLECTION_SUMMARY Genomic Medicine at King Faisal Specialist Hospital and Research Center CO:COLLECTION_SUMMARY (KFSHRC). The samples were collected from MCADD newborns (n=14) and healthy CO:COLLECTION_SUMMARY newborns (controls) (n=14). These newborns were age- and gender-matched. The CO:COLLECTION_SUMMARY inclusion criteria for the patient group included newborns positively diagnosed CO:COLLECTION_SUMMARY with only MCADD through the newborn screening program’s platform. For the CO:COLLECTION_SUMMARY control group, the inclusion criteria were healthy, gender-and age-match CO:COLLECTION_SUMMARY newborns. Also, newborns with less than a month were included as the average age CO:COLLECTION_SUMMARY of MCADD newborns was 15.3 days, and healthy newborns were 11 days. Any DBS CO:COLLECTION_SUMMARY samples collected from newborns diagnosed with other IMD or older than a month CO:COLLECTION_SUMMARY were excluded. CO:COLLECTION_PROTOCOL_FILENAME MCAD_biological samples CO:SAMPLE_TYPE Blood (plasma) CO:STORAGE_CONDITIONS -20℃ #TREATMENT TR:TREATMENT_SUMMARY no treatment #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites Extraction The metabolites were extracted as reported before with SP:SAMPLEPREP_SUMMARY modification (43). In detail, one punch, a size of 3.2 mm, was collected from SP:SAMPLEPREP_SUMMARY each DBS sample and transferred into a 96-well plate for metabolite extraction. SP:SAMPLEPREP_SUMMARY Metabolite extraction was performed by adding 250 ul extraction solvent SP:SAMPLEPREP_SUMMARY (20:40:40) (H2O: ACN: MeOH) to each well with agitation for 2 hours at room SP:SAMPLEPREP_SUMMARY temperature. Subsequently, sample extracts were dried using SpeedVac (Thermo SP:SAMPLEPREP_SUMMARY Fischer, Christ, Germany). The dried samples were reconstituted in 100 ul of 50% SP:SAMPLEPREP_SUMMARY A: B mobile phase. (A: 0.1% Formic acid in H2O, B: 0.1% FA in 50% ACN: MeOH). SP:SAMPLEPREP_SUMMARY Additional punches were taken for quality control (QC) from the project samples SP:SAMPLEPREP_SUMMARY to maintain the instrument performance. SP:SAMPLEPREP_PROTOCOL_FILENAME Metabolites extraction #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Metabolomics analysis was explored using the Waters Acquity UPLC system coupled CH:CHROMATOGRAPHY_SUMMARY with a Xevo G2-S QTOF mass spectrometer equipped with an electrospray ionization CH:CHROMATOGRAPHY_SUMMARY source (ESI) (43,44). In detail, the extracted metabolites were chromatographed CH:CHROMATOGRAPHY_SUMMARY using an ACQUITY UPLC using XSelect (100×2.1mm 2.5 μm) column (Waters Ltd., CH:CHROMATOGRAPHY_SUMMARY Elstree, UK), the mobile phase composed of 0.1% formic acid in dH2O as solvent A CH:CHROMATOGRAPHY_SUMMARY and solvent B consists of 0.1% formic acid in 50% ACN: MeOH. A gradient elution CH:CHROMATOGRAPHY_SUMMARY schedule was run as follows: 0-16 min 95- 5% A, 16-19 min 5% A, 19-20 min 5-95% CH:CHROMATOGRAPHY_SUMMARY A, 20-22 min 95- 95% A, at 300 μL/min flow rate. MS spectra were acquired under CH:CHROMATOGRAPHY_SUMMARY positive and negative electrospray ionization modes (ESI+, ESI-). MS conditions CH:CHROMATOGRAPHY_SUMMARY were as follows: source temperature was 150◦C, the desolvation temperature was CH:CHROMATOGRAPHY_SUMMARY 500◦C (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 0 CH:CHROMATOGRAPHY_SUMMARY and 10-50 V, respectively, in MSE mode. The mass spectrometer was calibrated CH:CHROMATOGRAPHY_SUMMARY with sodium formate in 100–1200 Da. Data were collected in continuum mode with CH:CHROMATOGRAPHY_SUMMARY Masslynx™ V4.1 (Waters Technologies, Milford, MA., USA) workstation. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity UPLC XSelect HSS C18 (100 × 2.1mm, 2.5um) CH:SOLVENT_A 0.1% formic acid in dH2O CH:SOLVENT_B 0.1% formic acid in 50% MeOH and ACN 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:COLUMN_TEMPERATURE 55 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Xevo-G2-S MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE 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:MS_RESULTS_FILE ST002557_AN004213_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END