#METABOLOMICS WORKBENCH ReemAlMalki91_20230411_101738 DATATRACK_ID:3858 STUDY_ID:ST002560 ANALYSIS_ID:AN004219 PROJECT_ID:PR001652 VERSION 1 CREATED_ON April 14, 2023, 8:53 am #PROJECT PR:PROJECT_TITLE Hydroxylated acylcarnitines as potential biomarkers for VLCADD newborn patients PR:PROJECT_TITLE in Saudi Arabia PR:PROJECT_TYPE newborn screening PR:PROJECT_SUMMARY Very long acylcarnitine dehydrogenase deficiency (VLCADD) is an inherited PR:PROJECT_SUMMARY metabolic disorder related to fatty acid β-oxidation. It is characterized by PR:PROJECT_SUMMARY genetic mutations in ACADVL gene and accumulations of acylcarnitines. VLCADD can PR:PROJECT_SUMMARY be developed in the neonatal period or during adulthood. Certain diagnostic PR:PROJECT_SUMMARY approaches are used to confirm the diagnosis of VLCADD including genetic PR:PROJECT_SUMMARY sequencing and newborn bloodspot screening (NBS). The last two approaches have PR:PROJECT_SUMMARY shown some limitations such as VUS with genetic sequencing and false positive or PR:PROJECT_SUMMARY negative results in NBS. Therefore, there are demands for additional diagnostic PR:PROJECT_SUMMARY tools for VLCADD. Since VLCADD is associated with disrupted metabolism, PR:PROJECT_SUMMARY untargeted metabolomics, which is an analytical technique used to detect a PR:PROJECT_SUMMARY large-scale profiling of metabolites in biological samples, could be a useful PR:PROJECT_SUMMARY tool for diagnosis. We hypothesized that VLCADD newborns patients may exhibit a PR:PROJECT_SUMMARY unique metabolic profile and biomarkers compared to healthy newborns. Untargeted PR:PROJECT_SUMMARY metabolomics approach was conducted using liquid chromatography-mass PR:PROJECT_SUMMARY spectrometry (LC-MS) to measure the global metabolites in DBS cards collected PR:PROJECT_SUMMARY from VLCADD newborns (n=15) and healthy controls (n=15). Metabolite extraction PR:PROJECT_SUMMARY was performed and followed by LC-MS analysis. Multivariate and univariate PR:PROJECT_SUMMARY analyses were used to analyze the metabolomics data, and pathway and biomarker PR:PROJECT_SUMMARY analyses were also performed on the significantly endogenous identified PR:PROJECT_SUMMARY metabolites. A moderate T-test was used for statistical analysis with no PR:PROJECT_SUMMARY correction, and the cutoff was (p-value ≤ 0.05 and Fold Change 1.5). VLCADD PR:PROJECT_SUMMARY newborns had 2012 significantly dysregulated metabolites compared to healthy PR:PROJECT_SUMMARY newborns. 58 endogenous metabolites were upregulated while 148 endogenous PR:PROJECT_SUMMARY metabolites were downregulated. Pathway analyses showed phenylalanine, tyrosine, PR:PROJECT_SUMMARY and tryptophan biosynthesis as the most affected pathway. Potential metabolic PR:PROJECT_SUMMARY biomarker for VLCADD was 3,4-dihydroxytetradecanoylcarnitine with an area under PR:PROJECT_SUMMARY the curve (AUC) of 1, was in the top-15 biomarker list with the highest p-value PR:PROJECT_SUMMARY and FC, suggesting its high possibility to be used for diagnosis. However, PR:PROJECT_SUMMARY validation experiments of the biomarker is needed in following-up studies to PR:PROJECT_SUMMARY ensure its accuracy and reliability to be used as a VLCADD marker in the PR:PROJECT_SUMMARY clinical practice. PR:INSTITUTE King Faisal Specialist Hospital and Research Centre (KFSHRC) PR:LAST_NAME AlMalki PR:FIRST_NAME Reem PR:ADDRESS Zahrawi Street, Al Maather, Riyadh 11211, Saudi Arabia PR:EMAIL 439203044@student.ksu.edu.sa PR:PHONE 0534045397 #STUDY ST:STUDY_TITLE Hydroxylated acylcarnitines as potential biomarkers for VLCADD newborn patients ST:STUDY_TITLE in Saudi Arabia ST:STUDY_SUMMARY Very long acylcarnitine dehydrogenase deficiency (VLCADD) is an inherited ST:STUDY_SUMMARY metabolic disorder related to fatty acid β-oxidation. It is characterized by ST:STUDY_SUMMARY genetic mutations in ACADVL gene and accumulations of acylcarnitines. VLCADD can ST:STUDY_SUMMARY be developed in the neonatal period or during adulthood. Certain diagnostic ST:STUDY_SUMMARY approaches are used to confirm the diagnosis of VLCADD including genetic ST:STUDY_SUMMARY sequencing and newborn bloodspot screening (NBS). The last two approaches have ST:STUDY_SUMMARY shown some limitations such as VUS with genetic sequencing and false positive or ST:STUDY_SUMMARY negative results in NBS. Therefore, there are demands for additional diagnostic ST:STUDY_SUMMARY tools for VLCADD. Since VLCADD is associated with disrupted metabolism, ST:STUDY_SUMMARY untargeted metabolomics, which is an analytical technique used to detect a ST:STUDY_SUMMARY large-scale profiling of metabolites in biological samples, could be a useful ST:STUDY_SUMMARY tool for diagnosis. We hypothesized that VLCADD newborns patients may exhibit a ST:STUDY_SUMMARY unique metabolic profile and biomarkers compared to healthy newborns. Untargeted ST:STUDY_SUMMARY metabolomics approach was conducted using liquid chromatography-mass ST:STUDY_SUMMARY spectrometry (LC-MS) to measure the global metabolites in DBS cards collected ST:STUDY_SUMMARY from VLCADD newborns (n=15) and healthy controls (n=15). Metabolite extraction ST:STUDY_SUMMARY was performed and followed by LC-MS analysis. Multivariate and univariate ST:STUDY_SUMMARY analyses were used to analyze the metabolomics data, and pathway and biomarker ST:STUDY_SUMMARY analyses were also performed on the significantly endogenous identified ST:STUDY_SUMMARY metabolites. A moderate T-test was used for statistical analysis with no ST:STUDY_SUMMARY correction, and the cutoff was (p-value ≤ 0.05 and Fold Change 1.5). VLCADD ST:STUDY_SUMMARY newborns had 2012 significantly dysregulated metabolites compared to healthy ST:STUDY_SUMMARY newborns. 58 endogenous metabolites were upregulated while 148 endogenous ST:STUDY_SUMMARY metabolites were downregulated. Pathway analyses showed phenylalanine, tyrosine, ST:STUDY_SUMMARY and tryptophan biosynthesis as the most affected pathway. Potential metabolic ST:STUDY_SUMMARY biomarker for VLCADD was 3,4-dihydroxytetradecanoylcarnitine with an area under ST:STUDY_SUMMARY the curve (AUC) of 1, was in the top-15 biomarker list with the highest p-value ST:STUDY_SUMMARY and FC, suggesting its high possibility to be used for diagnosis. However, ST:STUDY_SUMMARY validation experiments of the biomarker is needed in following-up studies to ST:STUDY_SUMMARY ensure its accuracy and reliability to be used as a VLCADD marker in the ST:STUDY_SUMMARY clinical practice. 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 #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_VLCAD_18453492 Factor:VLCAD patient RAW_FILE_NAME=DR_Rajaa_VLCAD_18453492 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_19538077 Factor:VLCAD patient RAW_FILE_NAME=DR_Rajaa_VLCAD_19538077 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_17600969 Factor:VLCAD patient RAW_FILE_NAME=DR_Rajaa_VLCAD_17600969 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_18816714 Factor:VLCAD patient RAW_FILE_NAME=DR_Rajaa_VLCAD_18816714 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_19451880 Factor:VLCAD patient RAW_FILE_NAME=DR_Rajaa_VLCAD_19451880 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_21307430 Factor:VLCAD patient RAW_FILE_NAME=DR_Rajaa_VLCAD_21307430 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_21329779 Factor:VLCAD patient RAW_FILE_NAME=DR_Rajaa_VLCAD_21329779 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_12719767 Factor:VLCAD patient RAW_FILE_NAME=DR_Rajaa_VLCAD_12719767 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_17993106 Factor:VLCAD patient RAW_FILE_NAME=DR_Rajaa_VLCAD_17993106 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_19338020 Factor:VLCAD patient RAW_FILE_NAME=DR_Rajaa_VLCAD_19338020 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_20431022 Factor:VLCAD patient RAW_FILE_NAME=DR_Rajaa_VLCAD_20431022 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_20431846 Factor:VLCAD patient RAW_FILE_NAME=DR_Rajaa_VLCAD_20431846 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_208488028 Factor:VLCAD patient RAW_FILE_NAME=DR_Rajaa_VLCAD_208488028 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_MOH00024983310 Factor:VLCAD patient RAW_FILE_NAME=DR_Rajaa_VLCAD_MOH00024983310 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_MOH00027348749 Factor:VLCAD patient RAW_FILE_NAME=DR_Rajaa_VLCAD_MOH00027348749 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_20839145 Factor:VLCAD_Ctrl RAW_FILE_NAME=DR_Rajaa_VLCAD_20839145 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_20851208 Factor:VLCAD_Ctrl RAW_FILE_NAME=DR_Rajaa_VLCAD_20851208 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_21369944 Factor:VLCAD_Ctrl RAW_FILE_NAME=DR_Rajaa_VLCAD_21369944 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_20864956 Factor:VLCAD_Ctrl RAW_FILE_NAME=DR_Rajaa_VLCAD_20864956 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_21442034 Factor:VLCAD_Ctrl RAW_FILE_NAME=DR_Rajaa_VLCAD_21442034 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_21741272 Factor:VLCAD_Ctrl RAW_FILE_NAME=DR_Rajaa_VLCAD_21741272 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_19534121 Factor:VLCAD_Ctrl RAW_FILE_NAME=DR_Rajaa_VLCAD_19534121 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_20462989 Factor:VLCAD_Ctrl RAW_FILE_NAME=DR_Rajaa_VLCAD_20462989 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_21608083 Factor:VLCAD_Ctrl RAW_FILE_NAME=DR_Rajaa_VLCAD_21608083 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_21730762 Factor:VLCAD_Ctrl RAW_FILE_NAME=DR_Rajaa_VLCAD_21730762 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_21753790 Factor:VLCAD_Ctrl RAW_FILE_NAME=DR_Rajaa_VLCAD_21753790 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_19534501 Factor:VLCAD_Ctrl RAW_FILE_NAME=DR_Rajaa_VLCAD_19534501 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_20830418 Factor:VLCAD_Ctrl RAW_FILE_NAME=DR_Rajaa_VLCAD_20830418 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_20975207 Factor:VLCAD_Ctrl RAW_FILE_NAME=DR_Rajaa_VLCAD_20975207 SUBJECT_SAMPLE_FACTORS - DR_Rajaa_VLCAD_21390005 Factor:VLCAD_Ctrl RAW_FILE_NAME=DR_Rajaa_VLCAD_21390005 #COLLECTION CO:COLLECTION_SUMMARY Biological samples DBS samples were obtained from the metabolomics section in CO:COLLECTION_SUMMARY the Center for Genomic Medicine at King Faisal Specialist Hospital and Research CO:COLLECTION_SUMMARY Center (KFSHRC). The samples were collected from VLCADD newborns (n=15) and CO:COLLECTION_SUMMARY healthy newborns (controls) (n=15). These newborns were age- and gender-matched. CO:COLLECTION_SUMMARY The inclusion criteria for the patient group included newborns positively CO:COLLECTION_SUMMARY diagnosed with only VLCADD through the newborn screening program’s platform. CO:COLLECTION_SUMMARY For the 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 VLCADD newborns was 6.2 days, and healthy newborns were 5.6 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 VLCAD_biological_samples.docx CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY No treatment #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The metabolites were extracted as reported before with modification (43). In SP:SAMPLEPREP_SUMMARY detail, one punch, a size of 3.2 mm, was collected from each DBS sample and SP:SAMPLEPREP_SUMMARY transferred into a 96-well plate for metabolite extraction. Metabolite SP:SAMPLEPREP_SUMMARY extraction was performed by adding 250 ul extraction solvent (20:40:40) (H2O: SP:SAMPLEPREP_SUMMARY ACN: MeOH) to each well with agitation for 2 hours at room temperature. SP:SAMPLEPREP_SUMMARY Subsequently, sample extracts were dried using SpeedVac (Thermo Fischer, Christ, SP:SAMPLEPREP_SUMMARY Germany). The dried samples were reconstituted in 100 ul of 50% A: B mobile SP:SAMPLEPREP_SUMMARY phase. (A: 0.1% Formic acid in H2O, B: 0.1% FA in 50% ACN: MeOH). Additional SP:SAMPLEPREP_SUMMARY punches were taken for quality control (QC) from the project samples to maintain SP:SAMPLEPREP_SUMMARY the instrument performance. SP:SAMPLEPREP_PROTOCOL_FILENAME Metabolites_Extraction.docx #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 AN:ANALYSIS_PROTOCOL_FILE LC-MS_Metabolomics_VLCAD.docx #MS MS:INSTRUMENT_NAME Waters Xevo-G2-S MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE 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 ST002560_AN004219_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END