A False-Positive Case of Methylmalonic Aciduria by Tandem Mass Spectrometry Newborn Screening Dependent on Maternal Malnutrition in Pregnancy

Methylmalonic Acidurias (MMAs) are a group of inborn errors of metabolism (IEMs), specifically of propionate catabolism characterized by gastrointestinal and neurometabolic manifestations resulting from a deficiency in the function of methylmalonyl-CoA mutase, methylmalonyl-CoA epimerase, and cobalamin metabolism. In Expanded Newborn Screening (NBS), increased levels of propionylcarnitine (C3) and/or of its ratios by MS/MS analysis of dried blood spots (DBS) samples are suggestive for either Propionic Acidemia or MMAs. C3 elevation is not considered a specific marker for these disorders, resulting in high false-positive rates. The use of analyte ratios improves specificity, but it still cannot resolve the diagnostic issue. Second-tier testing are strongly recommended as confirmation of primary NBS results and for a differential diagnosis. LC-MS/MS analysis allows the quantification of more specific markers of the disorder. Here, we report the case of a newborn with a suspected MMA at Expanded NBS and at second-tier test. Given the urgent situation, in-depth diagnostic investigations were performed. Further investigations surprisingly revealed a Vitamin B12 deficiency due to a maternal malnutrition during pregnancy. This case emphasized that metabolic alterations at NBS may not only be influenced by genome and related to IEMs, but also to external factors and to maternal conditions.

Stock solution IS1 was prepared by dissolving internal standards in water/acetonitrile 80:20 with 0.4% formic acid to obtain a final concentration of 500 μM for the three ISs. Stock solution IS2 was prepared by diluting 1:50 the IS1 solution with water to obtain a final concentration of 10 μM for the three ISs. Internal standards stock solutions IS1 and IS2 were stored at +4°C. Working solution IS3 was prepared freshly by adding 1 M Dithiothreitol (DTT, purchased from Sigma-Aldrich) to IS2 solution diluted 1:10 with acetonitrile/water 70:30 with 0.5% formic acid. Calibrators and Quality Controls (QCs) were made by spotting onto filter paper some drops of whole blood from healthy donors fortified with different proportions of two standard solutions at a concentration of 10 mM and 1 mM, respectively. Calibrators and QCs were prepared following the indications in Supplementary Tables S3. For hcy, mma and mca quantification by LC-MS/MS analysis, 250 μL of working solution IS3 were added to two 3.2 mm-DBS disks of sample, calibrators and QCs. Each sample was gently mixed (20°C, 60 min) in a Thermomixer (Eppendorf®). The supernatant was transferred into a new 1.5 mL tube and dried in a SpeedVac. The residue was then reconstituted with 100 µL of 3N HCl in n-Butanol (purchased from Sigma-Aldrich) and mixed in a Thermomixer (65°C, 15 min). The sample was dried once again in a SpeedVac, then the residue was reconstituted with 100 µL of water, briefly centrifuged and the supernatant was transferred into polypropylene vial (provided by Waters Corporation). The vials were finally placed in the system autosampler for the LC-MS/MS analysis.
The LC-MS/MS system consisted of an ACQUITY UPLC I-Class/Xevo TQD IVD tandem quadrupole mass spectrometer (Waters Corporation, Milford, MA, USA). The system operated in positive electrospray ionization mode using TargetLynx XS software (Waters Corporation, Milford, MA, USA). 5 μL were injected into the ion source and the run time was 9 minutes, injection-toinjection. For UPLC analysis, the ACQUITY UPLC BEH C18 2.1 x 50mm column with ACQUITY UPLC BEG C18 VanGuard pre-column was used. The mobile phase comprised a binary solvent system: H2O (Solvent A) and ACN (Solvent B), both containing 0.1% formic acid. The initial solvent composition, 95% A and 5% B, was maintained for 1.0 minute. The flow gradient profile involved the following steps: increasing from the initial conditions to 90% B within 5.0 min, holding for 1.0 min before coming back to 95% A. The flow rate was 0,5 mL/min and the column was maintained to 40°C. An example of chromatogram is shown in Supplementary Figure S1.
Functions 1-3 in Supplementary Table S4 summarize all the parameters refer to the Multiple Reaction Monitoring (MRM) experiments created for each analyte.
All the information regarding the method reproducibility, accuracy and precision are detailed in Supplementary material Tables S5-S6.

Routinely Newborn Screening Analysis and Second-tier Testing
Dried blood spot (DBS) samples for NBS are punched out into 3.2 mm-disks to perform a flow injection-tandem mass spectrometry analysis (FIA-MS/MS) for the detection of 36 IEMs, including AAs, urea cycle, organic acid and fatty acid oxidation disorders. Actually, four 3.2 mm DBS disks are used to test by immunofluorimetric assays congenital hypothyroidism (CH), cystic fibrosis (CF), galactosemia and biotinidase deficiency, respectively, and the fifth DBS disk is employed for FIA-MS/MS analysis. For the latter, the DBS disk (of approximately 3-3.2 μL whole blood) is extracted for the determination of 14 AAs, 35 acylcarnitines (ACs), free carnitine and succinylacetone, by using the NeoBase 2 Non-Derivatized MSMS Kit (Perkin Elmer Life and Analytical Sciences, Turku, Finland. The FIA-MS/MS system consists of an Acquity UPLC I-Class coupled to a Xevo TQD tandem quadrupole mass spectrometer (Waters Corporation, Milford, MA, USA). The system operates in positive electrospray ionization mode by multiple reaction monitoring (MRM) acquisition. 10 μL are injected into the ion source and the run time is 1.1 min, injection-to-injection. Data are finally processed by MassLynx V4.2 and NeoLynx Software (Waters Corp.).

Second-tier test for the quantification of methylmalonic acid, methylcitric acid and homocysteine by LC-MS/MS
For C3 second tier test, the DBS sample is punched out twice into final diameter disks of approximately 3.2 mm, using an automatic puncher. Two DBS disks (equivalent to approximately 6-6.4 μL whole blood) are extracted for the determination of mma, mca and hcy by LC-MS/MS ( Figure  S1). Details of C3 second-tier analysis by LC-MS/MS are fully reported in in Table S3-S6.