Tandem mass spectrometry-based newborn screening correctly identifies individuals with very long-chain acyl-CoA dehydrogenase deficiency (VLCADD). However, a great number of healthy individuals present with identical acylcarnitine profiles during catabolism in the first three days of life. We routinely perform an enzyme activity assay as confirmation analysis in newborns identified by screening. Whereas VLCAD residual activities of less than 10% are clearly diagnostic and indicate patients at risk of clinical disease, the clinical relevance of higher residual activities is unclear. In this study we assess the molecular basis in 34 individuals with residual activities of 10-50%. We identify two pathogenic mutations in patients that result in residual activities as high as 22%, while individuals with residual activities of 25-50% either present with a heterozygous or no mutation in the VLCAD gene. In addition, confirmed heterozygous parents present with residual activities as low as 32%.In conclusion, we identify individuals with 2 pathogenic mutations and those with only one heterozygous mutation in the residual activity range of 20-30%. Whereas heterozygosity is generally regarded as clinically irrelevant, identification of 2 VLCAD mutations leads to precautions in the management of the children. Based on our data we anticipate that individuals with a residual enzyme activity >20% present with a biochemical phenotype but likely remain asymptomatic throughout life. Studies in greater patient numbers are needed to correlate residual activities >10% with the genotype and the outcome.

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BackgroundNewborn screening for medium- and very long-chain acyl-CoA dehydrogenase (MCAD and VLCAD, respectively) deficiency, using acylcarnitine profiling with tandem mass spectrometry, has increased the number of patients with fatty acid oxidation disorders due to the identification of additional milder, and so far silent, phenotypes. However, especially for VLCADD, the acylcarnitine profile can not constitute the sole parameter in order to reliably confirm disease. Therefore, we developed a new liquid chromatography tandem mass spectrometry (LC-MS/MS) method to rapidly determine both MCAD- and/or VLCAD-activity in human lymphocytes in order to confirm diagnosis.MethodologyLC-MS/MS was used to measure MCAD- or VLCAD-catalyzed production of enoyl-CoA and hydroxyacyl-CoA, in human lymphocytes.Principal FindingsVLCAD activity in controls was 6.95±0.42 mU/mg (range 1.95 to 11.91 mU/mg). Residual VLCAD activity of 4 patients with confirmed VLCAD-deficiency was between 0.3 and 1.1%. Heterozygous ACADVL mutation carriers showed residual VLCAD activities of 23.7 to 54.2%. MCAD activity in controls was 2.38±0.18 mU/mg. In total, 28 patients with suspected MCAD-deficiency were assayed. Nearly all patients with residual MCAD activities below 2.5% were homozygous 985A>G carriers. MCAD-deficient patients with one other than the 985A>G mutation had higher MCAD residual activities, ranging from 5.7 to 13.9%. All patients with the 199T>C mutation had residual activities above 10%.ConclusionsOur newly developed LC-MS/MS method is able to provide ample sensitivity to correctly and rapidly determine MCAD and VLCAD residual activity in human lymphocytes. Importantly, based on measured MCAD residual activities in correlation with genotype, new insights were obtained on the expected clinical phenotype.