How mass spectrometry revolutionized newborn screening.

This article offers a personal account of a remarkable journey spanning over 30 years of applied mass spectrometry in a clinical setting. It begins with the author's inspiration from a clinician's story of rescuing a child from near death with a revolutionary therapeutic intervention. Motivated by this experience, the author delved into the field of chemistry and mass spectrometry to solve an analytical challenge. The breakthrough came with the development of the first front-line diagnostic test performed by MS/MS, which focused on analyzing acylcarnitines to detect and diagnose inherited disorders related to fatty acid and branched-chain amino acid catabolism. Building upon this success, the author expanded the application of the method to dried blood spots, incorporating additional analytical components such as essential amino acids. The result was a groundbreaking multiplex assay capable of screening newborns for more than 30 inherited metabolic conditions with just one test. This novel approach laid the foundation for a targeted metabolomics platform that facilitated the identification of new animal models of metabolic disease through screening the offspring of genetically modified adults. The development and utilization of MS/MS with UPLC has led to the creation of new assays for biomarkers of metabolic disease, benefiting both the diagnosis and therapeutic monitoring of these conditions. The article provides compelling examples from the author's laboratory, highlighting the value and vast applications of these methods in the field of metabolic disease research.

Quantification of Glycosaminoglycans in Urine by Isotope-Dilution Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry.

Mucopolysaccharidoses (MPSs) are complex lysosomal storage disorders that result in the accumulation of glycosaminoglycans (GAGs) in urine, blood, and tissues. Lysosomal enzymes responsible for GAG degradation are defective in MPSs. GAGs including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS) are disease-specific biomarkers for MPSs. This article describes a stable isotope dilution-tandem mass spectrometric method for quantifying CS, DS, and HS in urine samples. The GAGs are methanolyzed to uronic or iduronic acid-N-acetylhexosamine or iduronic acid-N-sulfo-glucosamine dimers and mixed with internal standards derived from deuteriomethanolysis of GAG standards. Specific dimers derived from HS, DS, and CS are separated by ultra-performance liquid chromatography (UPLC) and analyzed by electrospray ionization tandem mass spectrometry (MS/MS) using selected reaction monitoring for each targeted GAG product and its corresponding internal standard. This UPLC-MS/MS GAG assay is useful for identifying patients with MPS types I, II, III, VI, and VII. © 2023 Wiley Periodicals LLC. Basic Protocol: Urinary GAG analysis by ESI-MS/MS Support Protocol 1: Prepare calibration samples Support Protocol 2: Preparation of stable isotope-labeled internal standards Support Protocol 3: Preparation of quality controls for GAG analysis in urine Support Protocol 4: Optimization of the methanolysis time Support Protocol 5: Measurement of the concentration of methanolic HCl.

Addition of MPS-II to the Recommended Uniform Screening Panel in the United States.

It has recently been announced that the Secretary of the U.S. Department of Health and Human Services has approved the recommendation by the Advisory Committee on Heritable Disorders in Newborns and Children (ACHDNC) to add mucopolysaccharidosis type II (MPS-II, Hunter Syndrome) to the recommended uniform screening panel (RUSP) in the United States [...].

The Editor's Choice for Issue 4, Volume 7.

Dear Readers: welcome to the Editor's Choice for Volume 7, Issue 4 of the International Journal for Newborn Screening [...].

Successful Implementation of Expanded Newborn Screening in the Philippines Using Tandem Mass Spectrometry.

Newborn bloodspot screening (NBS) began as a research project in the Philippines in 1996 and was mandated by law in 2004. The program initially included screening for five conditions, with a sixth added in 2012. As screening technology and medical knowledge have advanced, NBS programs in countries with developed economies have also expanded, not only in the number of newborns screened but also in the number of conditions included in the screening. Various approaches have been taken regarding selection of conditions to be screened. With limited resources, low- and middle-income countries face significant challenges in selecting conditions for screening and in implementing sustainable screening programs. Building on expansion experiences in the U.S. and data from California on Filipinos born and screened there, the Philippine NBS program has recently completed its expansion to include 29 screening conditions. This report focuses on those conditions detectable through tandem mass spectrometry. Expanded screening was implemented in a stepwise fashion across the seven newborn screening laboratories in the Philippines. A university-based biochemical genetics laboratory provides confirmatory testing. Follow-up care for confirmed cases is monitored and provided through the NBS continuity clinics across the archipelago. Pre-COVID-19 pandemic, the coverage was 91.6% but dropped to 80.4% by the end of 2020 due to closure of borders between cities, provinces, and islands.

The Editor's Choice for Issue 1, Volume 7.

Dear Readers: welcome to the second issue of the Editor's Choice, continuing the tradition started by Peter Schielen's appraisal of Issue 4 of Volume 6 of IJNS, published in this issue [...].

Capillary Electrophoresis-High Resolution Mass Spectrometry for Measuring In Vivo Arginine Isotope Incorporation in Alzheimer's Disease Mouse Models.

Immune-based metabolic reprogramming of arginine utilization in the brain contributes to the neuronal pathology associated with Alzheimer's disease (AD). To enable our long-term goals of differentiation of AD mouse model genotypes, ages, and sexes based on activity of this pathway, we describe here the novel dosing (using uniformly labeled (13C615N4) arginine) and analysis methods using capillary electrophoresis high-resolution accurate-mass mass spectrometry for isotope tracing of metabolic products of arginine. We developed a pseudoprimed infusion-dosing regimen, using repeated injections, to achieve a steady state of uniformly labeled arginine in 135-195 min post bolus dose. Incorporation of stable isotope labeled carbon and nitrogen from uniformly labeled arginine into a host of downstream metabolites was measured in vivo in mice using serially sampled dried blood spots from the tail. In addition to the dried blood spot time course samples, total isotope incorporation into arginine-related metabolites was measured in the whole brain and plasma after 285 min. Preliminary demonstration of the technique identified differences isotope incorporation in arginine metabolites between male and female mice in a mouse-model of sporadic Alzheimer's disease (APOE4/huNOS2). The technique described herein will permit arginine pathway activity differentiation between mouse genotypes, ages, sexes, or drug treatments in order to elucidate the contribution of this pathway to Alzheimer's disease.

Digital Microfluidics in Newborn Screening for Mucopolysaccharidoses: A Progress Report.

Newborn screening (NBS) for mucopolysaccharidosis type I (MPS I, Hurler syndrome) is currently conducted in about two-fifths of the NBS programs in the United States and in a few other countries. Screening is performed by measurement of residual activity of the enzyme alpha-l-iduronidase in dried blood spots using either tandem mass spectrometry or digital microfluidic fluorometry (DMF). In this article, we focus on the development and practical experience of using DMF to screen for MPS I in the USA. By means of their responses to a questionnaire, we determined for each responding program that is screening for MPS I using DMF the screen positive rate, follow-up methods, and classification of confirmed cases as either severe or attenuated. Overall, the results show that at the time of reporting, over 1.3 million newborns in the US were screened for MPS I using DMF, 2094 (0.173%) of whom were screen positive. Of these, severe MPS I was confirmed in five cases, attenuated MPS I was confirmed in two cases, and undetermined phenotype was reported in one case. We conclude that DMF is an effective and economical method to screen for MPS I and recommend second-tier testing owing to high screen positive rates. Preliminary results of NBS for MPS II and MPS III using DMF are discussed.

Comparison of dermatan sulfate and heparan sulfate concentrations in serum, cerebrospinal fluid and urine in patients with mucopolysaccharidosis type I receiving intravenous and intrathecal enzyme replacement therapy.

AIMS: To validate a liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the measurement of glycosaminoglycans (GAGs) in plasma and serum. To establish plasma, cerebrospinal fluid (CSF) and urine reference intervals. To compare GAGs in serum with that in urine and CSF from patients with MPS I. METHODS: Dermatan sulfate (DS), heparan sulfate (HS), and chondroitin sulfate (CS) in serum/plasma, urine and CSF were methanolysed into dimers and analyzed using pseudo isotope dilution UPLC-MS/MS assay. Serum, CSF and urine DS and HS were quantified for 11 patients with mucopolysaccharidosis (MPS) type I before and after treatment with Aldurazyme® (laronidase) enzyme replacement therapy (ERT). RESULTS: The method showed acceptable imprecision and recovery for the quantification of serum/plasma CS, DS, and HS. The serum, urine, and CSF DS and HS concentrations were reduced after 26 weeks of ERT in 4 previously untreated patients. Serum DS and HS concentrations normalized in some patients, and were mildly elevated in others after ERT. In contrast, urine and CSF DS and HS values remained elevated above the reference ranges. Compared with serum GAGs, urine and CSF DS and HS were more sensitive biomarkers for monitoring the ERT treatment of patients with MPS I.

Acylcarnitine metabolomic profiles inform clinically-defined major depressive phenotypes.

BACKGROUND: Acylcarnitines have important functions in mitochondrial energetics and ß-oxidation, and have been implicated to play a significant role in metabolic functions of the brain. This retrospective study examined whether plasma acylcarnitine profiles can help biochemically distinguish the three phenotypic subtypes of major depressive disorder (MDD): core depression (CD+), anxious depression (ANX+), and neurovegetative symptoms of melancholia (NVSM+). METHODS: Depressed outpatients (n = 240) from the Mayo Clinic Pharmacogenomics Research Network were treated with citalopram or escitalopram for eight weeks. Plasma samples collected at baseline and after eight weeks of treatment with citalopram or escitalopram were profiled for short-, medium- and long-chain acylcarnitine levels using AbsoluteIDQ®p180-Kit and LC-MS. Linear mixed effects models were used to examine whether acylcarnitine levels discriminated the clinical phenotypes at baseline or eight weeks post-treatment, and whether temporal changes in acylcarnitine profiles differed between groups. RESULTS: Compared to ANX+, CD+ and NVSM+ had significantly lower concentrations of short- and long-chain acylcarnitines at both baseline and week 8. In NVSM+, the medium- and long-chain acylcarnitines were also significantly lower in NVSM+ compared to ANX+. Short-chain acylcarnitine levels increased significantly from baseline to week 8 in CD+ and ANX+, whereas medium- and long-chain acylcarnitines significantly decreased in CD+ and NVSM+. CONCLUSIONS: In depressed patients treated with SSRIs, ß-oxidation and mitochondrial energetics as evaluated by levels and changes in acylcarnitines may provide the biochemical basis of the clinical heterogeneity of MDD, especially when combined with clinical characteristics.

Evaluation of X-Linked Adrenoleukodystrophy Newborn Screening in North Carolina.

Importance: X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal genetic disorder in which an accumulation of very long-chain fatty acids leads to inflammatory demyelination in the central nervous system and to adrenal cortex atrophy. In 2016, X-ALD was added to the US Recommended Uniform Screening Panel. Objective: To evaluate the performance of a single-tier newborn screening assay for X-ALD in North Carolina. Design, Setting, and Participants: This diagnostic screening study was of all newborn dried blood spot specimens received in the North Carolina State Laboratory of Public Health between January 2 and June 1, 2018, excluding specimens of insufficient quantity or quality. A total of 52 301 specimens were screened for X-ALD using negative ionization high-performance liquid chromatography tandem mass spectrometry to measure C24:0- and C26:0-lysophosphatidylcholine concentrations. Sanger sequencing of the adenosine triphosphate-binding cassette subfamily D member 1 (ABCD1) gene was performed on screen-positive specimens. Exposures: A medical and family history, newborn physical examination, sequencing of ABCD1 on dried blood spot samples, and plasma analysis of very long-chain fatty acids were obtained for all infants with screen-positive results. Main Outcomes and Measures: The prevalence of X-ALD in North Carolina and the positive predictive value and false-positive rate for the first-tier assay were determined. Results: Of 52 301 infants tested (47.8% female, 50.6% male, and 1.7% other or unknown sex), 12 received screen-positive results. Of these 12 infants, 8 were confirmed with a genetic disorder: 3 male infants with X-ALD, 3 X-ALD-heterozygous female infants, 1 female infant with a peroxisome biogenesis disorder, and 1 female infant with Aicardi-Goutières syndrome. Four infants were initially classified as having false-positives results, including 3 female infants who were deemed unaffected and 1 male infant with indeterminate results on confirmatory testing. The positive predictive value for X-ALD or other genetic disorders for the first-tier assay was 67%, with a false-positive rate of 0.0057%. Conclusions and Relevance: This newborn screening pilot study reported results on 2 lysophosphatidylcholine analytes, identifying 3 male infants with X-ALD, 3 X-ALD-heterozygous female infants, and 3 infants with other disorders associated with increased very long-chain fatty acids. These results showed successful implementation in a public health program with minimal risk to the population. The findings will support other state laboratories planning to implement newborn screening for X-ALD and related disorders.

Glycocalyx breakdown is increased in African children with cerebral and uncomplicated falciparum malaria.

Cerebral malaria (CM) from Plasmodium falciparum infection is associated with endothelial dysfunction and parasite sequestration. The glycocalyx (GCX), a carbohydrate-rich layer lining the endothelium, is crucial in vascular homeostasis. To evaluate the role of its loss in the pathogenesis of pediatric CM, we measured GCX degradation in Tanzanian children with World Health Organization-defined CM (n = 55), uncomplicated malaria (UM; n = 20), and healthy controls (HCs; n = 25). Urine GCX breakdown products [glycosaminoglycans (GAGs)] were quantified using dimethylmethylene blue (DMMB) and liquid chromatography-tandem mass spectrometry assays. DMMB-GAG and mass spectrometry (MS)-GAG (g/mol creatinine) were increased in CM and UM compared with HCs (P < 0.001), with no differences in DMMB-GAG and MS-GAG between CM and UM children or between those with and without a fatal outcome. In CM survivors, urinary GCX DMMB-GAG normalized by d 3. After adjusting for disease severity, DMMB-GAG was significantly associated with parasitemia [partial correlation coefficient (Pcorr) = 0.34; P = 0.01] and plasma TNF (Pcorr = 0.26; P = 0.04) and inversely with plasma and urine NO oxidation products [Pcorr = -0.31 (P = 0.01) and Pcorr = -0.26 (P = 0.03), respectively]. GCX breakdown is increased in children with falciparum malaria, with similar elevations in CM and UM. Endothelial GCX degradation may impair endothelial NO production, exacerbate adhesion-molecule expression, exposure, and parasite sequestration, and contribute to malaria pathogenesis.-Yeo, T. W., Bush, P. A., Chen, Y., Young, S. P., Zhang, H., Millington, D. S., Granger, D. L., Mwaikambo, E. D., Anstey, N. M., Weinberg, J. B. Glycocalyx breakdown is increased in African children with cerebral and uncomplicated falciparum malaria.

The North Carolina Experience with Mucopolysaccharidosis Type I Newborn Screening.

OBJECTIVE: To evaluate the performance of a 2-tiered newborn screening method for mucopolysaccharidosis type I (MPS I) in North Carolina. STUDY DESIGN: The screening algorithm included a flow injection analysis-tandem mass spectrometry assay as a first-tier screening method to measure α-L-iduronidase (IDUA) enzyme activity and Sanger sequencing of the IDUA gene on dried blood spots as a second-tier assay. The screening algorithm was revised to incorporate the Collaborative Laboratory Integrated Reports, an analytical interpretive tool, to reduce the false-positive rate. A medical history, physical examination, IDUA activity, and urinary glycosaminoglycan (GAG) analysis were obtained on all screen-positive infants. RESULTS: A total of 62 734 specimens were screened with 54 screen-positive samples using a cut-off of 15% of daily mean IDUA activity. The implementation of Collaborative Laboratory Integrated Reports reduced the number of specimens that screened positive to 19 infants. Of the infants identified as screen-positive, 1 had elevated urinary GAGs and a homozygous pathogenic variant associated with the severe form of MPS I. All other screen-positive infants had normal urinary GAG analysis; 13 newborns had pseudodeficiency alleles, 3 newborns had variants of unknown significance, and 2 had heterozygous pathogenic variants. CONCLUSIONS: An infant with severe MPS I was identified and referred for a hematopoietic stem cell transplant. Newborn IDUA enzyme deficiency is common in North Carolina, but most are due to pseudodeficiency alleles in infants with normal urinary GAG analysis and no evidence of disease. The pilot study confirmed the need for second-tier testing to reduce the follow-up burden.

Glycocalyx Breakdown Is Associated With Severe Disease and Fatal Outcome in Plasmodium falciparum Malaria.

BACKGROUND: Interactions between the endothelium and infected erythrocytes play a major role in the pathogenesis of falciparum malaria, with microvascular dysfunction and parasite sequestration associated with worsening outcomes. The glycocalyx is a carbohydrate-rich layer that lines the endothelium, with multiple roles in vascular homeostasis. The role of the glycocalyx in falciparum malaria and the association with disease severity has not been investigated. METHODS: We prospectively enrolled Indonesian inpatients (aged ≥18 years) with severe (SM) or moderately severe (MSM) falciparum malaria, as defined by World Health Organization criteria, and healthy controls (HCs). On enrollment, blood and urine samples were collected concurrently with measurements of vascular nitric oxide (NO) bioavailability. Urine was assayed for glycocalyx breakdown products (glycosaminoglycans) using a dimethylmethylene blue (GAG-DMMB) and liquid chromatography-tandem mass spectrometry (GAG-MS) assay. RESULTS: A total of 129 patients (SM = 43, MSM = 57, HC=29) were recruited. GAG-DMMB and GAG-MS (g/mol creatinine) were increased in SM (mean, 95% confidence interval: 3.98, 2.44-5.53 and 6.82, 5.19-8.44) compared to MSM patients (1.78, 1.27-2.29 and 4.87, 4.27-5.46) and HCs (0.22, 0.06-0.37 and 1.24, 0.89-1.59; P < 0.001). In SM patients, GAG-DMMB and GAG-MS were increased in those with a fatal outcome (n = 3; median, interquartile range: 6.72, 3.80-27.87 and 12.15, 7.88-17.20) compared to survivors (n = 39; 3.10, 0.46-4.5 and 4.64, 2.02-15.20; P = 0.03). Glycocalyx degradation was significantly associated with parasite biomass in both MSM (r = 0.48, GAG-DMMB and r = 0.43, GAG-MS; P < 0.001) and SM patients (r = 0.47, P = 0.002 and r = 0.33, P = 0.04) and inversely associated with endothelial NO bioavailability. CONCLUSIONS: Increased endothelial glycocalyx breakdown is associated with severe disease and a fatal outcome in adults with falciparum malaria.

Kinetic and Cross-Sectional Studies on the Genesis of Hypoargininemia in Severe Pediatric Plasmodium falciparum Malaria.

The low bioavailability of nitric oxide (NO) and its precursor, arginine, contributes to the microvascular pathophysiology of severe falciparum malaria. To better characterize the mechanisms underlying hypoargininemia in severe malaria, we measured the plasma concentrations of amino acids involved in de novo arginine synthesis in children with uncomplicated falciparum malaria (UM; n = 61), children with cerebral falciparum malaria (CM; n = 45), and healthy children (HC; n = 109). We also administered primed infusions of l-arginine uniformly labeled with 13C6 and 15N4 to 8 children with severe falciparum malaria (SM; age range, 4 to 9 years) and 7 healthy children (HC; age range, 4 to 8 years) to measure the metabolic flux of arginine, hypothesizing that arginine flux is increased in SM. Using two different tandem mass spectrometric methods, we measured the isotopic enrichment of arginine in plasma obtained at 0, 60, 90, 120, 150, and 180 min during the infusion. The plasma concentrations of glutamine, glutamate, proline, ornithine, citrulline, and arginine were significantly lower in UM and CM than in HC (P ≤ 0.04 for all pairwise comparisons). Of these, glutamine concentrations were the most markedly decreased: median, 457 µM (interquartile range [IQR], 400 to 508 µM) in HC, 300 µM (IQR, 256 to 365 µM) in UM, and 257 µM (IQR, 195 to 320 µM) in CM. Arginine flux during steady state was not significantly different in SM than in HC by the respective mass spectrometric methods: 93.2 µmol/h/kg of body weight (IQR, 84.4 to 129.3 µmol/h/kg) versus 88.0 µmol/h/kg (IQR, 73.0 to 102.2 µmol/h/kg) (P = 0.247) by the two mass spectrometric methods in SM and 93.7 µmol/h/kg (IQR, 79.1 to 117.8 µmol/h/kg) versus 81.0 µmol/h/kg (IQR, 75.9 to 88.6 µmol/h/kg) (P = 0.165) by the two mass spectrometric methods in HC. A limited supply of amino acid precursors for arginine synthesis likely contributes to the hypoargininemia and NO insufficiency in falciparum malaria in children.

The Role of Technology in Newborn Screening.

This commentary traces the expansion of newborn screening for inherited metabolic disorders during the past 55 years, from the first simple test for phenylketonuria to the current panel of over 35 conditions. Emphasis is placed on the role played by technology and the contributions made by researchers in North Carolina.

Acetyl-l-carnitine deficiency in patients with major depressive disorder.

The lack of biomarkers to identify target populations greatly limits the promise of precision medicine for major depressive disorder (MDD), a primary cause of ill health and disability. The endogenously produced molecule acetyl-l-carnitine (LAC) is critical for hippocampal function and several behavioral domains. In rodents with depressive-like traits, LAC levels are markedly decreased and signal abnormal hippocampal glutamatergic function and dendritic plasticity. LAC supplementation induces rapid and lasting antidepressant-like effects via epigenetic mechanisms of histone acetylation. This mechanistic model led us to evaluate LAC levels in humans. We found that LAC levels, and not those of free carnitine, were decreased in patients with MDD compared with age- and sex-matched healthy controls in two independent study centers. Secondary exploratory analyses showed that the degree of LAC deficiency reflected both the severity and age of onset of MDD. Moreover, these analyses showed that the decrease in LAC was larger in patients with a history of treatment-resistant depression (TRD), among whom childhood trauma and, specifically, a history of emotional neglect and being female, predicted the decreased LAC. These findings suggest that LAC may serve as a candidate biomarker to help diagnose a clinical endophenotype of MDD characterized by decreased LAC, greater severity, and earlier onset as well as a history of childhood trauma in patients with TRD. Together with studies in rodents, these translational findings support further exploration of LAC as a therapeutic target that may help to define individualized treatments in biologically based depression subtype consistent with the spirit of precision medicine.

Digital microfluidics comes of age: high-throughput screening to bedside diagnostic testing for genetic disorders in newborns.

INTRODUCTION: Digital microfluidics (DMF) is an emerging technology with the appropriate metrics for application to newborn and high-risk screening for inherited metabolic disease and other conditions that benefit from early treatment. Areas covered: This review traces the development of electrowetting-based DMF technology toward the fulfillment of its promise to provide an inexpensive platform to conduct enzymatic assays and targeted biomarker assays at the bedside. The high-throughput DMF platform, referred to as SEEKER®, was recently authorized by the United States Food and Drug Administration to screen newborns for four lysosomal storage disorders (LSDs) and is deployed in newborn screening programs in the United States. The development of reagents and methods for LSD screening and results from screening centers are reviewed. Preliminary results from a more compact DMF device, to perform disease-specific test panels from small volumes of blood, are also reviewed. Literature for this review was sourced using principal author and subject searches in PubMed. Expert commentary: Newborn screening is a vital and highly successful public health program. DMF technology adds value to the current testing platforms that will benefit apparently healthy newborns with underlying genetic disorders and infants at-risk for conditions that present with symptoms in the newborn period.

Clinical heterogeneity of mitochondrial NAD kinase deficiency caused by a NADK2 start loss variant.

Mitochondrial NAD kinase deficiency (NADK2D, OMIM #615787) is a rare autosomal recessive disorder of NADPH biosynthesis that can cause hyperlysinemia and dienoyl-CoA reductase deficiency (DECRD, OMIM #616034). NADK2 deficiency has been reported in only three unrelated patients. Two had severe, unremitting disease; one died at 4 months and the other at 5 years of age. The third was a 10 year old female with CNS anomalies, ataxia, and incoordination. In two cases mutations in NADK2 have been demonstrated. Here, we report the fourth known case, a 15 year old female with normal intelligence and a mild clinical and biochemical phenotype presumably without DECRD. Her clinical symptoms, which are now stable, became evident at the age of 9 with the onset of decreased visual acuity, bilateral optic atrophy, nystagmus, episodic lower extremity weakness, peripheral neuropathy, and gait abnormalities. Plasma amino acid levels were within normal limits except for mean lysine and proline levels that were 3.7 and 2.5 times the upper limits of normal. Whole exome sequencing (WES) revealed homozygosity for a g.36241900 A>G p. Met1Val start loss mutation in the primary NADK2 transcript (NM_001085411.1) encoding the 442 amino acid isoform. This presumed hypomorphic mutation has not been previously reported and is absent from the v1000GP, EVS, and ExAC databases. Our patient's normal intelligence and stable disease expands the clinical heterogeneity and the prognosis associated with NADK2 deficiency. Our findings also clarify the mechanism underlying NADK2 deficiency and suggest that this disease should be ruled out in cases of hyperlysinemia, especially those with visual loss, and neurological phenotypes.

Current State of the Art of Newborn Screening for Lysosomal Storage Disorders.

Prospective full-population newborn screening for multiple lysosomal storage disorders (LSDs) is currently practiced in a few NBS programs, and several others are actively pursuing this course of action. Two platforms suitable for multiple LSD screening-tandem mass spectrometry (MS/MS) and digital microfluidic fluorometry (DMF)-are now commercially available with reagent kits. In this article, we review the methods currently used for prospective NBS for LSDs and objectively compare their workflows and the results from two programs in the United States that screen for the same four LSDs, one using MS/MS and the other DMF. The results show that the DMF platform workflow is simpler and generates results faster than MS/MS, enabling results reporting on the same day as specimen analysis. Furthermore, the performance metrics for both platforms while not identical, are broadly similar and do not indicate the superior performance of one method over the other. Results show a preponderance of inconclusive results for Pompe and Fabry diseases and for Hurler syndrome, due to genetic heterogeneity and other factors that can lead to low enzyme activities, regardless of the screening method. We conclude that either platform is a good choice but caution that post-analytical tools will need to be applied to improve the positive predictive value for these conditions.