Butyrylcarnitine Elevation in Newborn Screening: Reducing False Positives and Distinguishing between Two Rare Diseases through the Evaluation of New Ratios.

One of the main challenges of newborn screening programs, which screen for inherited metabolic disorders, is cutting down on false positives (FPs) in order to avoid family stresses, additional analyses, and unnecessary costs. False positives are partly caused by an insubstantial number of robust biomarkers in evaluations. Another challenge is how to distinguish between diseases which share the same primary marker and for which secondary biomarkers are just as highly desirable. Focusing on pathologies that involve butyrylcarnitine (C4) elevation, such as short-chain acylCoA dehydrogenase deficiency (SCADD) and isobutyrylCoA dehydrogenase deficiency (IBDD), we investigated the acylcarnitine profile of 121 newborns with a C4 increase to discover secondary markers to achieve two goals: reduce the FP rate and discriminate between the two rare diseases. Analyses were carried out using tandem mass spectrometry with whole blood samples spotted on filter paper. Seven new biomarkers (C4/C0, C4/C5, C4/C5DC\C6OH, C4/C6, C4/C8, C4/C14:1, C4/C16:1) were identified using a non-parametric ANOVA analysis. Then, the corresponding cut-off values were found and applied to the screening program. The seven new ratios were shown to be robust (p < 0.001 and p < 0.01, 0.0937 < ε2 < 0.231) in discriminating between FP and IBDD patients, FP and SCADD patients, or SCADD and IBDD patients. Our results suggest that the new ratios are optimal indicators for identifying true positives, distinguishing between two rare diseases that share the same primary biomarker, improving the predictive positive value (PPV) and reducing the false positive rate (FPR).

Neuroactive Amino Acid Profile in Autism Spectrum Disorder: Results from a Clinical Sample.

Biological bases of autism spectrum disorder (ASD) include both genetic and epigenetic causes. Patients with ASD show anomalies in the profile of certain plasma amino acids, including neuroactive amino acids. Monitoring plasma amino acids may be relevant for patient care and interventions. We evaluated the plasma amino acid profile in samples extracted from dry blood spots by electrospray ionization-tandem mass spectrometry. Fourteen amino acids and eleven amino acid ratios were examined in patients with ASD and intellectual disability (ID), and neurotypical control subjects (TD). The amino acid profile in the ASD group showed reduced levels of ornithine (p = 0.008), phenylalanine (p = 0.042) and tyrosine (p = 0.013). The statistically significant amino acid ratios were Leu+Val/Phe+Tyr (p = 0.002), Tyr/Leu (p = 0.007) and Val/Phe (p = 0.028), such differences remaining significant only in the comparison between ASD and TD. Finally, a positive correlation emerged between the score of the restricted and repetitive behavior on ADOS-2 and the citrulline levels in the ASD group (p = 0.0047). To conclude, patients with ASD may show a distinguishable metabolic profile useful for studying their metabolic pathways in order to develop screening tests and targeted therapies.

Expanded Newborn Screening in Italy Using Tandem Mass Spectrometry: Two Years of National Experience.

Newborn screening (NBS) for inborn errors of metabolism is one of the most advanced tools for secondary prevention in medicine, as it allows early diagnosis and prompt treatment initiation. The expanded newborn screening was introduced in Italy between 2016 and 2017 (Law 167/2016; DM 13 October 2016; DPCM 12-1-2017). A total of 1,586,578 infants born in Italy were screened between January 2017 and December 2020. For this survey, we collected data from 15 Italian screening laboratories, focusing on the metabolic disorders identified by tandem mass spectrometry (MS/MS) based analysis between January 2019 and December 2020. Aminoacidemias were the most common inborn errors in Italy, and an equal percentage was observed in detecting organic acidemias and mitochondrial fatty acids beta-oxidation defects. Second-tier tests are widely used in most laboratories to reduce false positives. For example, second-tier tests for methylmalonic acid and homocysteine considerably improved the screening of CblC without increasing unnecessary recalls. Finally, the newborn screening allowed us to identify conditions that are mainly secondary to a maternal deficiency. We describe the goals reached since the introduction of the screening in Italy by exchanging knowledge and experiences among the laboratories.

New ratio as a useful marker for early diagnosis of proximal urea cycle disorders.

BACKGROUND AND AIMS: Proximal urea cycle disorders (PUCDs) are not included in most newborn screening programs due to the lack of adequate markers to monitor. Failure to alter citrulline and glutamine levels, the prognostic markers commonly used, can results in high false negative. Therefore, new biomarkers, prognostic of PUCDs, are strongly desirable. MATERIALS AND METHODS: We used tandem mass spectrometry to analyze blood spot from PUCDs patients during their follow up in our referral center focusing on glutamine to glutamate (Gln/Glu) ratio. We reanalyzed the same specimens of three patients after two months and the specimen of a new patient with suspicious of PUCD disorder. RESULTS: Specimens of our patients shown a significant elevation of the ratio Gln/Glu compared to that of a healthy population (p < 0.05) as well as the specimens analyzed after two months, while the glutamine concentration dropped. New patient, showing high value of the ratio, was molecularly confirmed as PUCD patient. We further analyzed the blood spots from a neonatal population in order to fix a cut-off value and include it in a newborn screening panel. CONCLUSION: Our preliminary results suggest that the Gln/Glu ratio could be a very useful diagnostic marker, more stable over time than glutamine, which could improve the performance in early PUCDs identification.

Neonatal Screening on Tandem Mass Spectrometry as a Powerful Tool for the Reassessment of the Prevalence of Underestimated Diseases in Newborns and Their Family Members: A Focus on Short Chain Acyl-CoA Dehydrogenase Deficiency.

Early detection of disabling diseases, prior to clinical manifestations, is the primary goal of newborn screening (NS). Indeed, the required number of core and secondary conditions selected for screening panels is increasing in many countries. Furthermore, newborn screening can lead to diagnosis of maternal diseases such as vitamin B12 deficiency or 3-MethylcrotonylCoA-carboxylase deficiency (3MCC). NS became mandatory in Sicily in December 2017. Here we report NS data collected between December 2017 and April 2020. Our results show that tandem mass spectrometry is a powerful tool for discovery of underestimated disease in newborns and their family members. Our panel included short chain acyl-CoA dehydrogenase deficiency (SCADD). Here, we report that results of our investigation led to reassessment of SCADD prevalence in our population. The infant and adult patients diagnosed in our study had previously not shown overt symptoms.

Expanded Newborn Screening Using Tandem Mass Spectrometry: Seven Years of Experience in Eastern Sicily.

The expanded newborn screening for selected inborn errors of metabolism (IEM) in Sicily was introduced in 2007 by a Regional project entitled "Early detection of congenital metabolic diseases: expanded neonatal screening". It established two newborn screening laboratories, for Western and Eastern Sicily, which started their activity in 2011. Here we present the results of expanded screening (excluding phenylketonuria (PKU)) of the Eastern laboratory from January 2011 to December 2017. Our data highlight the importance of the expanded newborn screening as a basic health program to avoid the underestimation of rare diseases and the need of further investigations even when there are no textbook alterations of the metabolic profiles. We performed our analysis on dried blood spot by tandem mass spectrometry, according to Italian guidelines. A total of 196 samples from 60,408 newborns gave positive screening results (recall rate 0.32%) while 12 babies were true positive, including 2 newborns whose mothers resulted in being affected by a metabolic disease. The overall frequency of IEM found in the screening panel was 1:6041 (mothers excluded) or 1:5034 (mothers included). The introduction of MS/MS technology in Sicily has significantly increased the detection of inherited metabolic disorders, including those not previously covered, with a predictable improved outcome for several disorders.