Metabolite Identification Using Infrared Ion Spectroscopy – Novel Biomarkers for Pyridoxine-Dependent Epilepsy

Untargeted LC-MS based metabolomics strategies are being increasingly applied in metabolite screening for a wide variety of medical conditions. The long-standing “grand challenge” in the utilization of this approach is metabolite identification – confidently determining the chemical structures of m/z-detected unknowns. Here, we use a novel workflow based on the detection of molecular features of interest by high-throughput untargeted LC-MS analysis of patient body fluids combined with targeted molecular identification of those features using infrared ion spectroscopy (IRIS), effectively providing diagnostic IR fingerprints for mass-isolated targets. A significant advantage of this approach is that in silico predicted IR spectra of candidate chemical structures can be used to suggest the molecular structure of unknown features, thus mitigating the need for the synthesis of a broad range of physical reference standards. Pyridoxine dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine metabolism, resulting from a mutation in the ALDH7A1 gene that leads to an accumulation of toxic levels of α-aminoadipic semialdehyde (α-AASA), piperideine-6-carboxylate (P6C), and pipecolic acid in body fluids. While α-AASA and P6C are known biomarkers for PDE in urine, their instability makes them poor candidates for diagnostic analysis from blood, which would be required for application in newborn screening protocols. Here, we use combined untargeted metabolomics-IRIS to identify several new biomarkers for PDE-ALDH7A1 that can be used for diagnostic analysis in urine, plasma, and cerebrospinal fluids, and are compatible with analysis in dried blood spots for newborn screening. The identification of these novel metabolites has directly rendered novel insights in the pathophysiology of PDE-ALDH7A1.

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Newborn screening and molecular features of patients with multiple acyl-CoA dehydrogenase deficiency in Quanzhou, China

Journal of Pediatric Endocrinology and Metabolism ◽

10.1515/jpem-2020-0694 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Yiming Lin ◽

Weifeng Zhang ◽

Zhixu Chen ◽

Chunmei Lin ◽

Weihua Lin ◽

...

Keyword(s):

Mass Spectrometry ◽

Genetic Analysis ◽

Tandem Mass Spectrometry ◽

Newborn Screening ◽

Dehydrogenase Deficiency ◽

Late Onset ◽

False Negative ◽

Tandem Mass ◽

Lipid Storage Myopathy ◽

Molecular Features

Abstract Objectives Multiple acyl-CoA dehydrogenase deficiency (MADD) is an autosomal recessive disorder of fatty acid, amino acid and choline metabolism. Late-onset MADD is caused by ETFDH mutations and is the most common lipid storage myopathy in China. However, few patients with MADD have been identified through newborn screening (NBS). This study assessed the acylcarnitine profiles and molecular features of patients with MADD identified through NBS. Methods From January 2014 to June 2020, 479,786 newborns screened via tandem mass spectrometry were recruited for this study. Newborns with elevated levels of multiple acylcarnitines were recalled, those who tested positive in the reassessment were referred for genetic analysis. Results Of 479,786 newborns screened, six were diagnosed with MADD. The MADD incidence in the Chinese population was estimated to be 1:79,964. Initial NBS revealed five patients with typical elevations in the levels of multiple acylcarnitines; however, in one patient, acylcarnitine levels were in the normal reference range during recall. Notably, one patient only exhibited a mildly increased isovalerylcarnitine (C5) level at NBS. The patient with an atypical acylcarnitine profile was diagnosed with MADD by targeted gene sequencing. Six distinct ETFDH missense variants were identified, with the most common variant being c.250G>A (p.A84T), with an allelic frequency of 58.35 (7/12). Conclusions These findings revealed that it is easy for patients with MADD to go unidentified, as they may have atypical acylcarnitine profiles at NBS and the recall stage, indicating the value of genetic analysis for confirming suspected inherited metabolic disorders in the NBS program. Therefore, false-negative (FN) results may be reduced by combining tandem mass spectrometry (MS/MS) with genetic testing in NBS for MADD.

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Utility of measuring very long-chain fatty-acyl carnitines in dried blood spots for newborn screening of X-linked Adrenoleukodystrophy

Molecular Genetics and Metabolism Reports ◽

10.1016/j.ymgmr.2021.100720 ◽

2021 ◽

Vol 26 ◽

pp. 100720

Author(s):

Archana Natarajan ◽

Rita Christopher ◽

Shruti V. Palakuzhiyil ◽

Sadanandavalli Retnaswami Chandra

Keyword(s):

Newborn Screening ◽

Dried Blood Spots ◽

Fatty Acyl ◽

Blood Spots ◽

Long Chain

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Concurrent Confirmation and Differential Diagnosis of Congenital Adrenal Hyperplasia from Dried Blood Spots: Application of a Second-Tier LC-MS/MS Assay in a Cross-Border Cooperation for Newborn Screening

Hormone Research in Paediatrics ◽

10.1159/000439380 ◽

2015 ◽

Vol 84 (5) ◽

pp. 311-318 ◽

Cited By ~ 9

Author(s):

Péter Monostori ◽

Pál Szabó ◽

Otilia Marginean ◽

Csaba Bereczki ◽

Eszter Karg

Keyword(s):

Differential Diagnosis ◽

Congenital Adrenal Hyperplasia ◽

Newborn Screening ◽

Dried Blood Spots ◽

Adrenal Hyperplasia ◽

Blood Spots ◽

Cross Border ◽

Second Tier ◽

Cross Border Cooperation

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Mass Spectrometry but Not Fluorimetry Distinguishes Affected and Pseudodeficiency Patients in Newborn Screening for Pompe Disease

Clinical Chemistry ◽

10.1373/clinchem.2016.269027 ◽

2017 ◽

Vol 63 (7) ◽

pp. 1271-1277 ◽

Cited By ~ 20

Author(s):

Hsuan-Chieh Liao ◽

Min-Ju Chan ◽

Chia-Feng Yang ◽

Chuan-Chi Chiang ◽

Dau-Ming Niu ◽

...

Keyword(s):

Mass Spectrometry ◽

Newborn Screening ◽

Pompe Disease ◽

Late Onset ◽

Asian Population ◽

Dried Blood Spots ◽

Fluorimetric Assay ◽

Analytical Range ◽

Patient Referrals

Abstract BACKGROUND Deficiency of the lysosomal enzyme acid α-glucosidase (GAA) causes Pompe disease. Newborn screening for Pompe disease is ongoing, and improved methods for distinguishing affected patients from those with pseudodeficiency, especially in the Asian population, would substantially reduce the number of patient referrals for clinical follow-up. METHODS We measured the enzymatic activity of GAA in dried blood spots on newborn screening cards (DBS) using a tandem mass spectrometry (MS/MS) assay. The assay displayed a relatively large analytical range compared to the fluorimetric assay with 4-methylumbelliferyl-α-glucoside. DBS from newborns confirmed to have infantile-onset Pompe disease (IOPD, n = 11) or late-onset Pompe disease (LOPD) (n = 12) and those from patients bearing pseudodeficiency alleles with or without Pompe mutations, or Pompe disease carriers (n = 230) were studied. RESULTS With use of the MS/MS GAA assay in DBS, 96% of the pseudodeficiency newborns and all of the Pompe disease carriers were well separated from the IOPD and LOPD newborns. The fluorimetric assay separated <10% of the pseudodeficiencies from the IOPD/LOPD group. CONCLUSIONS The relatively large analytical range MS/MS GAA assay but not the fluorimetric assay in DBS provides a robust approach to reduce the number of referrals and should dramatically facilitate newborn screening of Pompe disease.

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Newborn screening for severe combined immunodeficiency: Evaluation of a commercial T-cell receptor excision circle-based method in Victorian dried blood spots

Journal of Paediatrics and Child Health ◽

10.1111/jpc.13659 ◽

2017 ◽

Vol 54 (1) ◽

pp. 14-19 ◽

Cited By ~ 3

Author(s):

Stephanie Richards ◽

James Pitt ◽

Sharon Choo

Keyword(s):

T Cell ◽

Newborn Screening ◽

T Cell Receptor ◽

Severe Combined Immunodeficiency ◽

Cell Receptor ◽

Dried Blood Spots ◽

Combined Immunodeficiency ◽

Blood Spots

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Determination of psychosine concentration in dried blood spots from newborns that were identified via newborn screening to be at risk for Krabbe disease

Clinica Chimica Acta ◽

10.1016/j.cca.2013.01.017 ◽

2013 ◽

Vol 419 ◽

pp. 73-76 ◽

Cited By ~ 44

Author(s):

Wei-Lien Chuang ◽

Josh Pacheco ◽

X. Kate Zhang ◽

Monica M. Martin ◽

Chad K. Biski ◽

...

Keyword(s):

At Risk ◽

Newborn Screening ◽

Dried Blood Spots ◽

Krabbe Disease ◽

Blood Spots

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Second-Tier Test for Quantification of Alloisoleucine and Branched-Chain Amino Acids in Dried Blood Spots to Improve Newborn Screening for Maple Syrup Urine Disease (MSUD)

Clinical Chemistry ◽

10.1373/clinchem.2007.098434 ◽

2008 ◽

Vol 54 (3) ◽

pp. 542-549 ◽

Cited By ~ 65

Author(s):

Devin Oglesbee ◽

Karen A Sanders ◽

Jean M Lacey ◽

Mark J Magera ◽

Bruno Casetta ◽

...

Keyword(s):

Amino Acids ◽

Newborn Screening ◽

Dried Blood Spots ◽

Branched Chain Amino Acids ◽

Maple Syrup ◽

Blood Spots ◽

Urine Disease ◽

Second Tier ◽

Branched Chain ◽

Positive Results

Abstract Background: Newborn screening for maple syrup urine disease (MSUD) relies on finding increased concentrations of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine by tandem mass spectrometry (MS/MS). d-Alloisoleucine (allo-Ile) is the only pathognomonic marker of MSUD, but it cannot be identified by existing screening methods because it is not differentiated from isobaric amino acids. Furthermore, newborns receiving total parenteral nutrition often have increased concentrations of BCAAs. To improve the specificity of newborn screening for MSUD and to reduce the number of diet-related false-positive results, we developed a LC-MS/MS method for quantifying allo-Ile. Methods: Allo-Ile and other BCAAs were extracted from a 3/16-inch dried blood spot punch with methanol/H2O, dried under nitrogen, and reconstituted into mobile phase. Quantitative LC-MS/MS analysis of allo-Ile, its isomers, and isotopically labeled internal standards was achieved within 15 min. To determine a reference interval for BCAAs including allo-Ile, we analyzed 541 dried blood spots. We also measured allo-Ile in blinded samples from 16 MSUD patients and 21 controls and compared results to an HPLC method. Results: Intra- and interassay imprecision (mean CVs) for allo-Ile, leucine, isoleucine, and valine ranged from 1.8% to 7.4%, and recovery ranged from 91% to 129%. All 16 MSUD patients were correctly identified. Conclusions: The LC-MS/MS method can reliably measure allo-Ile in dried blood spots for the diagnosis of MSUD. Applied to newborn screening as a second-tier test, it will reduce false-positive results, which produce family anxiety and increase follow-up costs. The assay also appears suitable for use in monitoring treatment of MSUD patients.

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Preliminary Proficiency Testing Results for Succinylacetone in Dried Blood Spots for Newborn Screening for Tyrosinemia Type I

Clinical Chemistry ◽

10.1373/clinchem.2009.133819 ◽

2009 ◽

Vol 55 (12) ◽

pp. 2207-2213 ◽

Cited By ~ 12

Author(s):

Barbara W Adam ◽

Timothy H Lim ◽

Elizabeth M Hall ◽

W Harry Hannon

Keyword(s):

Newborn Screening ◽

Proficiency Testing ◽

False Negative ◽

Dried Blood Spots ◽

Screening Tests ◽

Type I ◽

Primary Metabolite ◽

Screening Practices ◽

Tyrosinemia Type I ◽

Stable Performance

Abstract Background: Succinylacetone (SUAC) is the primary metabolite accumulated in tyrosinemia type I—an inborn error of metabolism that, if untreated, can cause death from liver failure during the first months of life. Newborn screening laboratories measure SUAC in dried blood spot (DBS) samples to detect asymptomatic tyrosinemia type I. We used panels of SUAC-enriched DBSs to compare and evaluate the performance of these screening tests. Methods: We prepared sets of DBS materials enriched with predetermined SUAC concentrations and distributed samples of these materials, along with a screening practices questionnaire, to laboratories that perform SUAC tests. We compared their reported SUAC concentrations and questionnaire responses to identify screening practices that affect SUAC test outcomes. Results: Data from 2 pilot surveys showed large differences among laboratories in SUAC recoveries, reproducible within-laboratory recoveries, and stable performance of the DBS materials. Results from 257 proficiency test analyses contained a total of 6 false-negative misclassifications. Reported recoveries of added SUAC ranged from 0 to >200%. Low-biased SUAC recoveries were associated with 1 method used by 5 laboratories. All laboratories that reported SUAC recoveries ≥100% used DBS matrix calibrators. Conclusions: The wide ranges of SUAC concentrations reported for pilot and proficiency testing specimens demonstrate a need to harmonize quantitative results among laboratories. Although DBS matrix calibrators are important for optimizing SUAC recoveries, the preparation of these calibrators is not standardized among laboratories. Certified DBS-based SUAC calibrators are needed for accuracy and harmonization.

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