Determination of methylmalonic acid, 2-methylcitric acid, and total homocysteine in dried blood spots by liquid chromatography–tandem mass spectrometry: A reliable follow-up method for propionylcarnitine-related disorders in newborn screening

Abstract

<jats:sec><jats:title>Objectives</jats:title><jats:p> Determination of methylmalonic acid, 2-methylcitric acid, and total homocysteine in dried blood spots by liquid chromatography–tandem mass spectrometry has usually been used as a second-tier test to improve performance of newborn screening for propionylcarnitine-related disorders. However, factors that potentially affect its detection results have not been investigated, and we aimed to evaluate these influencing factors and explore their potential utility in newborn screening and initial follow-up for propionylcarnitine-related disorders. </jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p> This study comprised a prospective group (1998 healthy infants, to establish cutoff values and investigate the influencing factors) and a retrospective group (804 suspected positive cases screened from 381, 399 newborns for propionylcarnitine-related disorders by tandem mass spectrometry, to evaluate the performance of newborn screening and initial follow-up). </jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p> Cutoff values for methylmalonic acid, 2-methylcitric acid, and total homocysteine were 2.12, 0.70, and 10.05 µmol/l, respectively. Concentration of methylmalonic acid, 2-methylcitric acid, and total homocysteine in dried blood spots is not impacted by sex, age, birth weight, gestational age, or dried blood spot storage time. A total of 75 of 804 cases were screened positive by combined tandem mass spectrometry and liquid chromatography–tandem mass spectrometry, thus eliminating 90% of the false positives without compromising sensitivity. Eighteen propionylcarnitine-related disorders were successfully identified, including one CblX case missed in the initial follow-up by tandem mass spectrometry. </jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p> Methylmalonic acid, 2-methylcitric acid, and total homocysteine detected in dried blood spots by liquid chromatography–tandem mass spectrometry is a reliable, specific, and sensitive approach for identifying propionylcarnitine-related disorders. We recommend this assay should be performed rather than tandem mass spectrometry in follow-up for propionylcarnitine-related disorders besides second-tier tests in newborn screening. </jats:p></jats:sec>

Application of a next-generation sequencing (NGS) panel in newborn screening efficiently identifies inborn disorders of neonates

“…If the parents choose to report carrier result and the infant identified as carrier, we can provide genetic counseling for them. The top genes such as PAH , SLC22A5 , ACADS and MUT were most frequently reported for carriers known to have high carrier frequency in reported study [ 46 ], which could verify the relative high incidence of associated inborn disorders [ 50 , 51 ]. Some high-frequency mutations of DUOX2, HBB , ATP7B gene in our study were not found in previous investigations.…”

“…Using NBGS, 4 cases were diagnosed with MMA, PCD and WD that had been missed by C-NBS. It has been reported that the determination of MMA by MS/MS has been affected by several factors, and one case was missed due to the concentrations of C3 and C3/C0 decrease with age [ 46 , 47 ]. However, unlike metabolites, genetic screening was sufficiently accurate and specific for inborn disorders because it does not vary with age, season or maternal status.…”

Validation of a targeted metabolomics panel for improved second‐tier newborn screening

“…DBS from a subset of true-positive (TP) and false-positive (FP) samples (Table S1) were blinded and processed in one analytic batch. For each DBS, a single 1/8-inch diameter circle was punched into a 96-deepwell plate, mixed with 100 μL of a DBS extraction solution containing internal standards (prepared as 90 mL methanol, 10 mL water, 20 μL formic acid, 40 μL methylmalonic acid-D 3 [1 mg/mL], 40 μL each of NSK-B-1 and NSK-B-G1, 50 μL of MSK-A2 amino acids mix, 8 μL ornithine 13 C 5 [5 mg/mL], 2 μL citrulline 13 C 5 [5 mg/mL], 2 μL dimethylglycine D 6 [1 mg/mL], 10 μL uridine 13 C 5 [1 mg/mL], 40 μL orotic acid D 2 [6 mM]) and vortexed for 15 min. Following centrifugation at 1800Âg for 30 sec, supernatants (80 μL) were transferred to a new 96-deepwell plate and evaporated under nitrogen to dryness, and then reconstituted with 100 μL of a diluent solution (80% water, 20% methanol, 0.1% formic acid v/v/ v).…”

“…7,8 In contrast to first-tier screening using direct injection analysis, second-tier tests often use liquid chromatography-tandem mass spectrometry (LC-MS/MS) to increase specificity by distinguishing isomeric compounds [9][10][11][12] and evaluating additional disease-related metabolites. 3,13 False-positive cases can also be reduced using postanalytic tools applied to first-tier screening data with the Collaborative Laboratory Integrated Reports (CLIR), [14][15][16] and more recently using machine learningbased methodologies. [17][18][19][20] However, the improvement in screening performance with these approaches varies widely among disorders on the Recommended Uniform Screening Panel (RUSP), suggesting that identification and clinical validation of additional markers could further improve screening performance.…”

Treatment of metabolic disorders using genomic technologies: Lessons from methylmalonic acidemia

“…14 Some NS programs have further implemented the addition of measuring methylmalonic acid and/or 2-methylcitrate as secondary markers. [15][16][17][18] In addition to the biochemical changes, patients with MMA have a pleiotropic clinical phenotype characterized by heterogeneous multisystemic disease manifestations including, but not limited to, recurrent metabolic instability, propensity to develop encephalopathy with hyperammonemia, failure to thrive, feeding intolerance, metabolic strokes of the basal ganglia, developmental delays, hypotonia, optic nerve atrophy, pancreatitis, osteopenia, cardiomyopathy, chronic kidney disease, poor feeding, and overall, an increased risk for early death, especially those with mut 0 MMA. [2][3][4][19][20][21][22] These clinical phenotypes are heterogeneous and can vary greatly between affected individuals.…”