Fatal pitfalls in newborn screening for mitochondrial trifunctional protein (MTP)/long-chain 3-Hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency

Background: Mitochondrial trifunctional protein (MTP) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency are long-chain fatty acid oxidation disorders with particularly high morbidity and mortality. Outcome can be favorable if diagnosed in time, prompting the implementation in newborn screening programs. Sporadic cases missed by the initial screening sample have been reported. However, little is known on pitfalls during confirmatory testing resulting in fatal misconception of the diagnosis. Results: We report a series of three patients with MTP and LCHAD deficiency, in whom diagnosis was missed by newborn screening, resulting in life-threatening metabolic decompensations within the first half year of life. Two of the patients showed elevated concentrations of primary markers C16-OH and C18:1-OH but were missed by confirmatory testing performed by the maternity clinic. A metabolic center was not consulted. Confirmatory testing consisted of analyses of acylcarnitines in blood and organic acids in urine, the finding of normal excretion of organic acids led to rejection and underestimation of the diagnosis, respectively. The third patient, a preterm infant, was not identified in the initial screening sample due to only moderate elevations of C16-OH and C18:1-OH and normal secondary markers and analyte ratios. Conclusion: Our observations highlight limitations of newborn screening for MTP/LCHAD deficiency. They confirm that analyses of acylcarnitines in blood and organic acids in urine alone are not suitable for confirmatory testing and molecular or functional analysis is crucial in diagnosing MTP/LCHAD deficiency. Mild elevations of primary biomarkers in premature infants need to trigger confirmatory testing. Our report underscores the essential role of specialized centers in confirming or ruling out diagnoses in suspicious screening results

A Rapid and Sensitive UPLC-MS/MS-Method for the Separation and Quantification of Branched-Chain Amino Acids from Dried Blood Samples of Patients with Maple Syrup Urine Disease (MSUD)

Newborn screening for MSUD is a special challenge since patients with MSUD can metabolically decompensate rapidly without adequate treatment within the first two weeks of life. However, the screening method does not detect the actual marker metabolite (alloisoleucine) specifically, but only as part of the group of the other isobaric amino acids leucine, isoleucine and hydroxyproline. We describe a sensitive and rapid second-tier UPLC-MS/MS method to determine branched-chain amino acids from the initial extraction of the screening sample. Quantification is based on a seven-point calibration curve. Reference ranges (mean ± SD in µmol/L) were determined from 179 normal, not pre-selected samples from the newborn screening: leucine: 72 ± 27; isoleucine: 37 ± 19; valine: 98 ± 46; hydroxyproline: 23 ± 13. The concentration of alloisoleucine was below the detection limit in about 55% of the cases, and the highest concentration was 1.9 µmol/L. In all 30 retrospectively studied screening samples from patients with confirmed MSUD the concentration of alloisoleucine was significantly increased. In 238 samples with false-positive newborn screening due to a significant increase in the combined concentration of leucine + isoleucine + alloisoleucine + hydroxyproline (400 to >4000 µmol/L), alloisoleucine was below 6.5 µmol/L (n = 57) or not detectable (n = 181). The application of this assay markedly reduces the false-positive rate and the associated anxiety and costs. It is also suitable for routinely monitoring blood spots of patients with MSUD

A Rapid and Sensitive UPLC-MS/MS-Method for the Separation and Quantification of Branched-Chain Amino Acids from Dried Blood Samples of Patients with Maple Syrup Urine Disease (MSUD)

Newborn screening for MSUD is a special challenge since patients with MSUD can metabolically decompensate rapidly without adequate treatment within the first two weeks of life. However, the screening method does not detect the actual marker metabolite (alloisoleucine) specifically, but only as part of the group of the other isobaric amino acids leucine, isoleucine and hydroxyproline. We describe a sensitive and rapid second-tier UPLC-MS/MS method to determine branched-chain amino acids from the initial extraction of the screening sample. Quantification is based on a seven-point calibration curve. Reference ranges (mean ± SD in µmol/L) were determined from 179 normal, not pre-selected samples from the newborn screening: leucine: 72 ± 27; isoleucine: 37 ± 19; valine: 98 ± 46; hydroxyproline: 23 ± 13. The concentration of alloisoleucine was below the detection limit in about 55% of the cases, and the highest concentration was 1.9 µmol/L. In all 30 retrospectively studied screening samples from patients with confirmed MSUD the concentration of alloisoleucine was significantly increased. In 238 samples with false-positive newborn screening due to a significant increase in the combined concentration of leucine + isoleucine + alloisoleucine + hydroxyproline (400 to >4000 µmol/L), alloisoleucine was below 6.5 µmol/L (n = 57) or not detectable (n = 181). The application of this assay markedly reduces the false-positive rate and the associated anxiety and costs. It is also suitable for routinely monitoring blood spots of patients with MSUD

Glutaric Aciduria Type I Missed by Newborn Screening: Report of Four Cases from Three Families

Glutaric aciduria type I (GA-1) is a rare autosomal-recessive disorder of the degradation of the amino acids lysine and tryptophan caused by mutations of the GCDH gene encoding glutaryl-CoA-dehydrogenase. Newborn screening (NBS) for this condition is based on elevated levels of glutarylcarnitine (C5DC) in dried blood spots (DBS). Here we report four cases from three families in whom a correctly performed NBS did not detect the condition. Glutarylcarnitine concentrations were either normal (slightly below) or slightly above the cut-off. Ratios to other acylcarnitines were also not persistently elevated. Therefore, three cases were defined as screen negative, and one case was defined as normal, after a normal control DBS sample. One patient was diagnosed after an acute encephalopathic crisis, and the other three patients had an insidious onset of the disease. GA-1 was genetically confirmed in all cases. Despite extensive efforts to increase sensitivity and specificity of NBS for GA-1, by adjusting cut-offs and introducing various ratios, the biological diversity still leads to false-negative NBS results for GA-1

Correction: Spenger et al. Glutaric Aciduria Type I Missed by Newborn Screening: Report of Four Cases from Three Families. Int. J. Neonatal Screen. 2021, 7, 32

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