Higher precision, first tier newborn screening for metachromatic leukodystrophy using 16:1-OH-sulfatide.

Newborn screening (NBS) for metachromatic leukodystrophy (MLD) is based on first-tier measurement of sulfatides in dried blood spots (DBS) followed by second-tier measurement of arylsulfatase A in the same DBS. This approach is very precise with 0-1 false positives per ∼30,000 newborns tested. Recent data reported here shows that the sulfatide molecular species with an α-hydroxyl, 16­carbon, mono-unsaturated fatty acyl group (16:1-OH-sulfatide) is superior to the original biomarker 16:0-sulfatide in reducing the number of first-tier false positives. This result is consistent across 4 MLD NBS centers. By measuring 16:1-OH-sulfatide alone or together with 16:0-sulfatide, the estimated false positive rate is 0.048% and is reduced essentially to zero with second-tier arylsulfatase A activity assay. The false negative rate is predicted to be extremely low based on the demonstration that 40 out of 40 newborn DBS from clinically-confirmed MLD patients are detected with these methods. The work shows that NBS for MLD is extremely precise and ready for deployment. Furthermore, it can be multiplexed with several other inborn errors of metabolism already tested in NBS centers worldwide.

Newborn screening for aromatic l-amino acid decarboxylase deficiency - Strategies, results, and implication for prevalence calculations.

BACKGROUND: Aromatic l-amino acid decarboxylase deficiency (AADCD) is a rare, autosomal-recessive neurometabolic disorder caused by variants in dopa decarboxylase (DDC) gene, resulting in a severe combined deficiency of serotonin, dopamine, norepinephrine, and epinephrine. Birth prevalence of AADCD varies by population. In pilot studies, 3-O-methyldopa (3-OMD) was shown to be a reliable biomarker for AADCD in high-throughput newborn screening (NBS) allowing an early diagnosis and access to gene therapy. To evaluate the usefulness of this method for routine NBS, 3-OMD screening results from the largest three German NBS centers were analyzed. METHODS: A prospective, multicenter (n = 3) NBS pilot study evaluated screening for AADCD by quantifying 3-OMD in dried blood spots (DBS) using tandem mass spectrometry (MS/MS). RESULTS: In total, 766,660 neonates were screened from January 2021 until June 2023 with 766,647 with unremarkable AADCD NBS (766,443 by 1st-tier analysis and 204 by 2nd-tier analysis) and 13 with positive NBS result recalled for confirmatory diagnostics (recall-rate about 1:59,000). Molecular genetic analysis confirmed AADCD (c.79C > T p.[Arg27Cys] in Exon 2 und c.215 A > C p.[His72Pro] in Exon 3) in one infant. Another individual was highly suspected with AADCD but died before confirmation (overall positive predictive value 0.15). False-positive results were caused by maternal L-Dopa use (n = 2) and prematurity (30th and 36th week of gestation, n = 2). However, in 63% (n = 7) the underlying etiology for false positive results remained unexplained. Estimated birth prevalence (95% confidence interval) was 1:766,660 (95% CI 1:775,194; 1:769,231) to 1:383,330 (95% CI 1:384,615; 1:383,142). The identified child remained asymptomatic until last follow up at the age of 9 months. CONCLUSIONS: The proposed screening strategy with 3-OMD detection in DBS is feasible and effective to identify individuals with AADCD. The estimated birth prevalence supports earlier estimations and confirms AADCD as a very rare disorder. Pre-symptomatic identification by NBS allows a disease severity adapted drug support to diminish clinical complications until individuals are old enough for the application of the gene therapy.

[Development of analytics in newborn screening-from the Guthrie card to genetics]. / Entwicklung der Analytik im Neugeborenen-Screening ­ Von der Guthrie-Karte zur Genetik.

For more than five decades, all newborns in Germany have been offered a screening examination for the early detection of congenital treatable diseases. Since its inception, about 35 million children have been screened in this way.Originally, screening exams only included early detection of phenylketonuria, which, without timely treatment, would lead to mental retardation that could no longer be corrected. The bacteriological Guthrie test allowed the detection of elevated concentrations of phenylalanine. The methods used today are the result of decades of development. They have been expanded to include tests to determine enzyme activities, immunoassays for the early detection of important hormonal disorders such as congenital hypothyroidism, and high-pressure liquid chromatography for the diagnosis of pathologic hemoglobins. The very sophisticated tandem mass spectrometry enables the simultaneous detection of amino acid and fatty acid compounds. Steroids can also be identified. The specificity can be further increased by combining tandem mass spectrometry with chromatographic pre-separation. In recent years, chemical-analytical analyses have been supplemented by genetic diagnostic methods such as quantitative or qualitative polymerase chain reaction (PCR).The current state of laboratory technology is by no means final. Both classical analytics and especially genetic methods are facing further rapid development. Although the expansion of screening is also a consequence of technical development, the inclusion of further congenital diseases is fundamentally dependent on the given therapy. But it is precisely here that many innovations are currently being investigated. Gene therapy is at the forefront of interest.

High Throughput Newborn Screening for Sickle Cell Disease - Application of Two-Tiered Testing with a qPCR-Based Primary screen.

BACKGROUND: Sickle cell disease (SCD) is a group of hemoglobinopathies with a common point mutation causing the production of sickle cell hemoglobin (HbS). In high-throughput newborn screening (NBS) for SCD, a two-step procedure is suitable, in which qPCR first pre-selects relevant samples that are differentiated by a second method. METHODS: Three NBS centers using qPCR-based primary screening for SCD performed a laboratory comparison. Methods using tandem MS or HPLC were used for differentiation. RESULTS: In a benchmarking test, 450 dried blood samples were analyzed. Samples containing HbS were detected as reliably by qPCR as by methods established for hemoglobinopathy testing. In a two-step screening approach, the 2nd-tier-analyses have to distinguish the carrier status from pathological variants. In nine months of regular screening, a total of 353,219 samples were analyzed using two-stage NBS procedures. The 1st-tier screening by qPCR reduced the number of samples for subsequent differentiation by>99.5%. Cases with carrier status or other variants were identified as inconspicuous while 78 cases with SCD were revealed. The derived incidence of 1:4,773, is in good agreement with previously published incidences. CONCLUSION: In high-throughput NBS for SCD, qPCR is suitable to focus 2nd-tier analyses on samples containing HbS, while being unaffected by factors such as prematurity or transfusions. The substantial reduction of samples numbers positively impacts resource conservation, sustainability, and cost-effectiveness. No false negative cases came to attention.

Tissue Specific Distribution and Activation of Sapindaceae Toxins in Horses Suffering from Atypical Myopathy.

Equine atypical myopathy is caused by hypoglycin A (HGA) and methylenecyclopropylglycine (MCPrG), the known protoxins of sycamore maple (Acer pseudoplatanus). Various tissues from five atypical myopathy cases were analyzed but only HGA was found. Whether deamination of MCPrG has already occurred in the intestine as the first stage of metabolization has not been investigated. Activation of the protoxins to methylenecyclopropylacetyl (MCPA)-CoA and methylenecyclopropylformyl (MCPF)-CoA, respectively, occurred mainly in the skeletal muscles, as evidenced by very high concentrations of MCPA-carnitine and MCPF-carnitine in this tissue. Inhibition of the acyl-CoA dehydrogenases of short- and medium-chain as well as branched-chain fatty acids by the toxins led to a strong increase in the corresponding acylcarnitines, again preferentially in skeletal muscles. An accumulation of the long-chain acylcarnitines beyond the level of the control samples could not be detected in the tissues. As a high amount of HGA was always found unmetabolized in the organs, we speculate that targeting the interruption of further metabolization might be a way to stop the progression of intoxication. Inhibition of the mitochondrial branched-chain amino acid aminotransferase, i.e., the first enzyme responsible for the activation of sycamore maple protoxins, could be a therapeutic approach.

Collaborative evaluation study on 18 candidate diseases for newborn screening in 1.77 million samples.

Analytical and therapeutic innovations led to a continuous but variable extension of newborn screening (NBS) programmes worldwide. Every extension requires a careful evaluation of feasibility, diagnostic (process) quality and possible health benefits to balance benefits and limitations. The aim of this study was to evaluate the suitability of 18 candidate diseases for inclusion in NBS programmes. Utilising tandem mass spectrometry as well as establishing specific diagnostic pathways with second-tier analyses, three German NBS centres designed and conducted an evaluation study for 18 candidate diseases, all of them inherited metabolic diseases. In total, 1 777 264 NBS samples were analysed. Overall, 441 positive NBS results were reported resulting in 68 confirmed diagnoses, 373 false-positive cases and an estimated cumulative prevalence of approximately 1 in 26 000 newborns. The positive predictive value ranged from 0.07 (carnitine transporter defect) to 0.67 (HMG-CoA lyase deficiency). Three individuals were missed and 14 individuals (21%) developed symptoms before the positive NBS results were reported. The majority of tested candidate diseases were found to be suitable for inclusion in NBS programmes, while multiple acyl-CoA dehydrogenase deficiency, isolated methylmalonic acidurias, propionic acidemia and malonyl-CoA decarboxylase deficiency showed some and carnitine transporter defect significant limitations. Evaluation studies are an important tool to assess the potential benefits and limitations of expanding NBS programmes to new diseases.

Dried blood spot eluates are suitable for testing of SARS-CoV-2 IgG antibodies targeting Spike protein 1 and Nucleocapsid protein.

Dried blood spots (DBS) provide easy handling and are thus a beneficial tool for data collection, e.g. for epidemiological studies. The suitability of DBS for the assessment of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was analyzed with regards to the use in future studies addressing seroprevalence in the population. 121 volunteers gave a venous blood sample and capillary blood samples on two DBS cards (PerkinElmer and Ahlstrom-Munksjö) via self-sampling under supervision. All samples were analyzed using the Anti-SARS-CoV-2 ELISA (IgG) and the Anti-SARS-CoV-2 NCP ELISA (IgG) from EUROIMMUN performed on the EUROIMMUN EUROLabWorkstation ELISA. Correlation coefficients between ELISA results based on the different sampling methods were calculated. Results of DBS analysis for SARS-CoV-2 IgG S1 and NCP highly correlated with the serum values (r = 0.96). In addition, the calculation of the phi coefficient showed no significant difference between the qualitative results of both sampling methods (rφ = 0.98-1.0). Further analysis of DBS eluates after prolonged storage of 6-8 h also showed a high correlation with serum results (r = 0.97 and r = 0.93, respectively). The study results indicate suitability of DBS for the analysis of antibodies against SARS-CoV-2 S1 and NCP. For DBS eluate, a stability of 6-8 h for measurement of SARS-CoV-2 antibodies can be assumed.

Methionine Adenosyltransferase I/III Deficiency Detected by Newborn Screening.

Methionine adenosyltransferase I/III deficiency is an inborn error of metabolism due to mutations in the MAT1A gene. It is the most common cause of hypermethioninemia in newborn screening. Heterozygotes are often asymptomatic. In contrast, homozygous or compound heterozygous individuals can develop severe neurological symptoms. Less than 70 cases with biallelic variants have been reported worldwide. A methionine-restricted diet is recommended if methionine levels are above 500−600 µmol/L. In this study, we report on a female patient identified with elevated methionine concentrations in a pilot newborn screening program. The patient carries a previously described variant c.1132G>A (p.Gly378Ser) in homozygosity. It is located at the C-terminus of MAT1A. In silico analysis suggests impaired protein stability by ß-turn disruption. On a methionine-restricted diet, her serum methionine concentration ranged between 49−605 µmol/L (median 358 µmol/L). Her clinical course was characterized by early-onset muscular hypotonia, mild developmental delay, delayed myelination and mild periventricular diffusion interference in MRI. At 21 months, the girl showed age-appropriate neurological development, but progressive diffusion disturbances in MRI. Little is known about the long-term outcome of this disorder and the necessity of treatment. Our case demonstrates that neurological symptoms can be transient and even patients with initial neurologic manifestations can show normal development under dietary management.

Sudden neonatal death in individuals with medium-chain acyl-coenzyme A dehydrogenase deficiency: limit of newborn screening.

Medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency is the most common disorder of mitochondrial ß-oxidation of fatty acids resulting in hypoketotic hypoglycemia, hepatopathy, and often fatal outcome in undiagnosed children. Introduction of tandem mass spectrometry-based newborn screening programs in the late 1990s has significantly reduced morbidity and mortality in MCAD deficiency; however, neonatal death in individuals with early disease manifestation and severe hypoglycemia may still occur. We describe the fatal disease course in eight newborns with MCAD deficiency, aiming to raise awareness for early clinical symptoms and the life-saving treatment, and promote systematic post-mortem protocols for biochemical and genetic testing, necessary for correct diagnosis and counselling of the family if unexpected death occurred in the neonatal period.Conclusion: Early newborn screening and awareness for clinical symptoms is lifesaving in MCAD deficiency, which may present with fatal neonatal crisis. Systematic post-mortem diagnostic protocols are needed for sudden neonatal deaths.

The First 4 Years - Outcome of Children Identified by Newborn Screening for CF in Germany.

BACKGROUND: Newborn screening (NBS) has been shown to improve cystic fibrosis (CF) disease course and has been widely implemented worldwide. This monocentric study compared children diagnosed by NBS vs. a cohort preceding the implementation of NBS in Germany in 2016 to evaluate ascribed benefits of NBS. METHODS: We compared all children with confirmed CF diagnosis (n=19, "NBS group") out of all children presenting with positive NBS at our center after implementation of NBS (n=100) to children diagnosed with CF at our center within 4 years before NBS implementation (n=29, "pre-NBS group") for outcomes of anthropometry, gastrointestinal and pulmonary disease manifestations and respiratory microbiology. RESULTS: Children diagnosed by NBS had a lower incidence of initial difficulty to thrive (15 vs. 41%) and showed higher mean z-scores for Body-Mass-Index (BMI), weight and length at diagnosis and during study period. Children in the pre-NBS group displayed higher proportions of oxygen-dependent pulmonary exacerbations (10 vs. 0%). They show a significantly lower amount of normal bacterial flora (p=0.005) along with a significantly higher number of throat swab cultures positive for Pseudomonas aeruginosa (p=0.0154) in the first year of life. Yet, pulmonary imaging did not reveal less pulmonary morbidity in the NBS group. CONCLUSIONS: Our results confirm that NBS for CF leads to earlier diagnosis and improves nutritional outcomes in early childhood. Although trajectories of structural lung damage at early age were unaffected by NBS, NBS positive CF patients at preschool age displayed less pulmonary exacerbations and pathological bacteria in throat swabs.

Nitisinone treatment during two pregnancies and breastfeeding in a woman with tyrosinemia type 1 - a case report.

OBJECTIVES: Tyrosinaemia type 1, an inherited disorder of tyrosine metabolism, is usually treated with a tyrosine-defined diet and since 2000 with nitisinone. So far, data about effects of nitisone during pregnancy and breastfeeding are rare. This is the first report of two pregnancies in a patient with tyrosinaemia type 1 while under treatment with nitisinone. CASE PRESENTATION: We here present a 20-year-old female patient with tyrisonemia type 1 receiving treatment with nitisinone and a tyrosine-defined diet since she was diagnosed with tyrosinaemia type 1 at the age of 18 months. During two pregnancies blood concentrations of tyrosine, succinylacetone and nitisinone were measured regularly. Neither infant has tyrosinaemia type 1 and both showed an initial increase in concentrations of tyrosine, succinylacetone and nitisinone. All three metabolites dropped within two weeks after birth. Both were exclusively breastfed for about two weeks. Both children show age-appropriate physical and mental development. CONCLUSIONS: Nitisinone therapy during pregnancy and the short breastfeeding period did not result in adverse events in our patient or her children. Regular assessments of tyrosine, succinylacetone and nitisinone should be made during pregnancy and the breastfeeding period in both the mother and the infant. For better understanding, in principle, all cases of pregnancy and breastfeeding with tyrosinemia type 1 should be assessed and followed to further evaluate the implications of tyrosinaemia type 1 and its treatment during pregnancy. Additionally, even though experience with breastfeeding is limited, medication with nitisinone is safe and there is no reason to consider breastfeeding unsafe or to not recommend it.

Severe Inhibition of Long-Chain Acyl-CoA Enoylhydratase (EC 4.2.1.74) in a Newborn Foal Suffering From Atypical Myopathy.

In horses, congenital defects of energy production from long-chain fatty acids have not been described so far. In contrast, inhibition of fatty acid degradation caused by the toxins hypoglycin A and methylenecyclopropylglycine from various maple species are observed frequently. These non-proteinogenic aminoacids are passed on placentally to fetuses or with collostrum or milk to newborn foals. Nevertheless, newborn foals become very rarely symptomatic. Vertical transmission apparently is not sufficient to induce clinical disease without a particular genetic constellation being present. One of these rare cases was investigated here using samples from a mare and her foal. Intoxication by hypoglycin A and methylenecyclopropylglycine is also of interest to human pathology, because these toxins have caused fatal poisonings after consumption of certain fruits many times, especially in children. Maple toxins, their metabolites and some short-chain acyl compounds were quantified by ultrahigh-pressure liquid chromatography/tandem mass spectrometry. An comprehensive spectrum of long-chain acylcarnitines was prepared using electrospray ionization tandem mass spectrometry. Organic acids and acylglycines were determined by gas chromatography mass spectrometry. For evaluation, results of other horses poisoned by maple material as well as unaffected control animals were used. In the serum of the foal, hypoglycin A was detected at a low concentration only. Toxin metabolites reached <3.5% of the mean of a comparison group of horses suffering from atypical myopathy. The spectrum of acylcarnitines indicated enzyme inhibition in short-chain and medium-chain regions typical of acer poisoning, but the measured concentrations did not exceed those previously found in clinically healthy animals after maple consumption. The values were not sufficient to explain the clinical symptoms. In contrast, a remarkably strong enrichment of tetradecenoylcarnitine and hexadecenoylcarnitine was observed. This proves a blockade of the long-chain enoyl-CoA hydratase (EC 4.2.1.74). Vertical transfer of maple toxins to a newborn foal is sufficient for induction of clinical disease only if there is an additional specific reactivity to the active toxins. This was found here in an inhibition of long-chain enoyl-CoA hydratase. Isolated dysfunction of this enzyme has not yet been reported in any species. Further studies are necessary to prove a specific genetic defect.

L2HGDH Missense Variant in a Cat with L-2-Hydroxyglutaric Aciduria.

A 7-month-old, spayed female, domestic longhair cat with L-2-hydroxyglutaric aciduria (L-2-HGA) was investigated. The aim of this study was to investigate the clinical signs, metabolic changes and underlying genetic defect. The owner of the cat reported a 4-month history of multiple paroxysmal seizure-like episodes, characterized by running around the house, often in circles, with abnormal behavior, bumping into obstacles, salivating and often urinating. The episodes were followed by a period of disorientation and inappetence. Neurological examination revealed an absent bilateral menace response. Routine blood work revealed mild microcytic anemia but biochemistry, ammonia, lactate and pre- and post-prandial bile acids were unremarkable. MRI of the brain identified multifocal, bilaterally symmetrical and T2-weighted hyperintensities within the prosencephalon, mesencephalon and metencephalon, primarily affecting the grey matter. Urinary organic acids identified highly increased levels of L-2-hydroxyglutaric acid. The cat was treated with the anticonvulsants levetiracetam and phenobarbitone and has been seizure-free for 16 months. We sequenced the genome of the affected cat and compared the data to 48 control genomes. L2HGDH, coding for L-2-hydroxyglutarate dehydrogenase, was investigated as the top functional candidate gene. This search revealed a single private protein-changing variant in the affected cat. The identified homozygous variant, XM_023255678.1:c.1301A>G, is predicted to result in an amino acid change in the L2HGDH protein, XP_023111446.1:p.His434Arg. The available clinical and biochemical data together with current knowledge about L2HGDH variants and their functional impact in humans and dogs allow us to classify the p.His434Arg variant as a causative variant for the observed neurological signs in this cat.

Detection of maple toxins in mare's milk.

BACKGROUND: Plants from the Sapindaceae family that are consumed by horses (maple) and humans (ackee and litchi) are known to contain the toxins hypoglycin A and methylenecyclopropylglycine which cause seasonally occurring myopathy in horses and entero-encephalopathic sickness in humans. Vertical transmission of these toxins from a mare to her foal has been described once. However the mare's milk was not available for analysis in this case. We investigated mare's milk in a similar case. OBJECTIVE: We hypothesized that hypoglycin A and methylenecyclopropylglycine, like other amino acids' are secreted into the milk. ANIMALS: Mare with atypical myopathy. METHODS: A sample of the mare's milk and 6 commercial horse milk samples were extracted with a methanolic standard solution and analyzed for hypoglycin A, methylenecyclopropylglycine, and metabolites using tandem mass spectrometry after column chromatographic separation. RESULTS: There were hypoglycin A (0.4 µg/L) and the associated metabolites methylenecyclopropylacetyl glycine and carnitine (18.5 and 24.6 µg/L) plus increased concentrations of several acylcarnitines in the milk. The milk also contained methylenecyclopropylformyl glycine and carnitine (0.8 and 60 µg/L). The latter substances were also detected in 1 of 6 commercial horse milk samples. CONCLUSIONS AND CLINICAL IMPORTANCE: Transmission of the maple toxins can occur through mare's milk. Vertical transmission of Sapindacea toxins might also have importance for human medicine, for example, after consumption of ackee or litchi.

Study on the Metabolic Effects of Repeated Consumption of Canned Ackee.

Ackee fruits (Blighia sapida), an important food source in some tropical countries, can be the cause of serious poisoning. Ackees contain hypoglycin A and methylenecyclopropylglycine. Experiments were undertaken by a volunteer to elucidate the metabolic details of poisoning. Rapid intestinal absorption of the toxins was followed by their slow degradation to methylenecyclopropylacetyl and methylenecyclopropylformyl conjugates. Impairment of the metabolism of branched chain amino acids and ß-oxidation of fatty acids was found. Reduced enzyme activities were observed for several days after ingestion. A defined dose of fruit material caused significantly higher concentrations of metabolites when consumed 24 h after a previous ingestion than when consumed only once. The accumulation of toxins, toxin metabolites, and products of the intermediate metabolism after repeated consumption may, at least partly, explain the high frequency of fatal cases observed during harvesting. No inhibition of enzymes that degrade long-chain acyl compounds was observed in the experiments.

Process adapted calibration improves fluorometric pH sensor precision in sophisticated fermentation processes.

Miniaturization and automation have become increasingly popular in bioprocess development in recent years, enabling rapid high-throughput screening and optimization of process conditions. In addition, advances in the bioprocessing industry have led to increasingly complex process designs, such as pH and temperature shifts, in microbial fed-batch fermentations for optimal soluble protein expression in a range of hosts. However, in order to develop an accurate scale-down model for bioprocess screening and optimization, small-scale bioreactors must be able to accurately reproduce these complex process designs. Monitoring methods, such as fluorometric-based pH sensors, provide elegant solutions for the miniaturization of bioreactors, however, previous research suggests that the intrinsic fluorescence of biomass alters the sigmoidal calibration curve of fluorometric pH sensors, leading to inaccurate pH control. In this article, we present results investigating the impact of biomass on the accuracy of a commercially available fluorometric pH sensor. Subsequently, we present our calibration methodology for more precise online measurement and provide recommendations for improved pH control in sophisticated fermentation processes.

A newborn screening approach to diagnose 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.

3-Hydroxy-3-methylglutaryl-coenzyme A lyase deficiency (HMGCLD) is a rare autosomal recessively inherited metabolic disorder. Patients suffer from avoidable neurologically devastating metabolic decompensations and thus would benefit from newborn screening (NBS). The diagnosis is currently made by measuring dry blood spot acylcarnitines (C5OH and C6DC) followed by urinary organic acid profiling for the differential diagnosis from several other disorders. Using untargeted metabolomics (reversed-phase UHPLC coupled to an Orbitrap Elite hybrid mass spectrometer) of plasma samples from 5 HMGCLD patients and 19 age-matched controls, we found 3-methylglutaconic acid and 3-hydroxy-3-methylglutaric acid, together with 3-hydroxyisovalerylcarnitine as the most discriminating metabolites between the groups. In order to evaluate the NBS potential of these metabolites we quantified the most discriminating metabolites from untargeted metabolomics in 23 blood spots from 4 HMGCLD patients and 55 controls by UHPLC tandem mass spectrometry. The results provide a tool for expanded NBS of HMGCLD using tandem mass spectrometry. Selected reaction monitoring transition 262/85 could be used in a first-tier NBS analysis to screen for elevated 3-hydroxyisovalerylcarnitine. In a positive case, a second-tier analysis of 3-hydroxy-3-methylglutaric acid and 3-methylglutaconic acid in a dry blood spot using UHPLC tandem mass spectrometry instruments confirms the diagnosis. In conclusion, we describe the identification of new diagnostic biomarkers for HMGCLD and their application in NBS in dry blood spots. By using second-tier testing, all patients with HMGCLD were unequivocally and correctly diagnosed.

Diagnosis of severe growth hormone deficiency in the newborn.

OBJECTIVE: Severe neonatal growth hormone deficiency (GHD) can cause recurrent hypoglycaemia. Early diagnosis is warranted. The aim of the study was to analyse the GH content in screening cards of 25 affected and 281 healthy newborns. PATIENTS AND MEASUREMENTS: A total of 110 screening cards from ill newborns were sent to us for measuring GH content by a highly sensitive GH ELISA. Clinical information was obtainable in 61 cases. Severe GHD was defined by the presence of recurrent hypoglycaemia with a significant pituitary malformation or two additional pituitary hormone deficiencies. Screening cards from 281 healthy newborns (34.0-37.9 weeks) were prospectively analysed. RESULTS: In 25 newborns (5 preterm), the definition of severe GHD was fulfilled based on recurrent hypoglycaemia in combination with malformation of the pituitary or midline structures in 21 cases and combined TSH and ACTH deficiency in four cases. The median GH concentration of those affected with severe GHD was 3.9 µg/L (range: 1.1-11.8), significantly below the previously reported reference range (P < .001). A GH concentration of 7 µg/L was confirmed as the cut-off for term newborns with the best accuracy (90.0% sensitivity and 98.7% specificity). The 95% reference range for healthy preterm newborns (n = 151) was 7.6-47.1 µg/L (median: 20.3 µg/L). CONCLUSIONS: A GH content <7.0 µg/L in the newborn screening card confirms severe GHD with high accuracy. In preterm newborns, the lower limit of the reference interval was 0.6 µg/L higher than in term newborns. The newborn screening card is a valuable source for the very early diagnosis of GH deficiency.

Inter-laboratory analytical improvement of succinylacetone and nitisinone quantification from dried blood spot samples.

BACKGROUND: Nitisinone is used to treat hereditary tyrosinemia type 1 (HT-1) by preventing accumulation of toxic metabolites, including succinylacetone (SA). Accurate quantification of SA during newborn screening is essential, as is quantification of both SA and nitisinone for disease monitoring and optimization of treatment. Analysis of dried blood spots (DBS) rather than plasma samples is a convenient method, but interlaboratory differences and comparability of DBS to serum/plasma may be issues to consider. METHODS: Eight laboratories with experience in newborn screening and/or monitoring of patients with HT-1 across Europe participated in this study to assess variability and improve SA and nitisinone concentration measurements from DBS by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Quantification of nitisinone from both DBS and plasma was performed to assess sample comparability. In addition, efforts to harmonize laboratory procedures of SA and nitisinone quantifications during 5 rounds of analysis are described. RESULTS: Nitisinone levels measured from DBS and plasma strongly correlated (R 2 = 0.93). Due to partitioning of nitisinone to the plasma, levels were higher in plasma by a factor of 2.34. In the initial assessment of laboratory performance, all had linear calibrations of SA and nitisinone although there was large inter-laboratory variability in actual concentration measurements. Subsequent analytical rounds demonstrated markedly improved spread and precision over previous rounds, an outcome confirmed in a final re-test round. CONCLUSION: The study provides guidance for the determination of nitisinone and SA from DBS and the interpretation of results in the clinic. Inter-laboratory analytical harmonization was demonstrated through calibration improvements.

Molecular based newborn screening in Germany: Follow-up for cystinosis.

BACKGROUND: Newborn screening (NBS) programs for treatable metabolic disorders have been enormously successful, but molecular-based screening has not been broadly implemented so far. METHODS: This prospective pilot study was performed within the German NBS framework. DNA, extracted from dried blood cards was collected as part of the regular NBS program. As cystinosis has a prevalence of only 1:100,000-1:200,000, a molecular genetic assay for detection of the SMN1 gene mutation with a higher prevalence was also included in the screening process, a genetic defect that leads to spinal muscular atrophy (SMA). First tier multiplex PCR was employed for both diseases. The cystinosis screening employed assays for the three most common CTNS mutations covering 75% of German patients; in case of heterozygosity for one of these mutations, samples were screened by next generation sequencing (NGS) of the CTNS exons for 101 CTNS mutations. A detection rate of 98.5% is predicted using this approach. RESULTS: Between January 15, 2018 and May 31, 2019, 257,734 newborns were screened in Germany for cystinosis. One neonate was diagnosed with cystinosis, consistent with the known incidence of the disease. No false positive or false negatives were detected so far. Screening, communication of findings to parents, and confirmation of diagnosis were accomplished in a multi-disciplinary setting. This program was accomplished with the cooperation of hospitals, physicians, and parents. In the neonate diagnosed with cystinosis, oral cysteamine treatment began on day 18. After 16 months of treatment the child has no clinical signs of renal tubular Fanconi syndrome. CONCLUSIONS: This pilot study demonstrates the efficacy of a molecular-based neonatal screening program for cystinosis using an existing national screening framework.