Search Results (20)

Currently, Dutch newborns are screened for tyrosinemia type 1 (TT1) using succinylacetone (SA) as the biomarker. Although the sensitivity of the test is high, a high number of false positives is observed. Here, the aim is to evaluate the current Dutch newborn-screening protocol and to assess alternatives, specifically the use of biomarkers that are already being measured, to increase the positive predictive value (PPV). TT1 screening was performed with the Revvity NeoBase assay between 2008 and 2017, and since 2018, the Revvity NeoBase 2 assay has been used. Data from 2018 to 2021 were used for evaluation. To simulate alternative screening protocols, these data were enriched with results of referrals from other periods and a false negative (FN) from 2010. In 2018–2021, 693,821 newborns were screened, resulting in 23 referrals, of whom two were TT1 patients. For this period, to date, no FN have been reported, resulting in a provisional sensitivity of 100%, a specificity of 99.997%, and a PPV and negative predictive value of 9% and 100%, respectively. To improve the PPV, we combined SA, tyrosine (tyr), tyr × SA and tyr/phenylalanine and achieved a PPV of 72% for this dataset without introducing FN in the original dataset. This illustrates that future screening for TT1 may benefit from the addition of these biomarkers. Full article

Newborn screening for Phenylketonuria enables early detection and timely treatment with a phenylalanine-restricted diet to prevent severe neurological impairment. Although effective and in use for 60 years, screening, diagnostic, and treatment practices still vary widely across countries and centers. To evaluate the Phenylketonuria newborn screening practices internationally, we designed a survey with questions focusing on the laboratory aspect of the screening system. We analyzed 24 completed surveys from 23 countries. Most participants used the same sampling age range of 48–72 h; they used tandem mass spectrometry and commercial non-derivatized kits to measure phenylalanine (Phe), and had non-negative cut-off values (COV) set mostly at 120 µmol/L of Phe. Participants mostly used genetic analysis of blood and detailed amino acid analysis from blood plasma as their confirmatory methods and set the COV for the initiation of dietary therapy at 360 µmol/L of Phe. There were striking differences in practice as well. While most participants reported a 48–72 h range for age at sampling, that range was overall quite diverse Screening COV varied as well. Additional screening parameters, e.g., the phenylalanine/tyrosine ratio were used by some participants to determine the screening result. Some participants included testing for tetrahydrobiopterin deficiency, or galactosemia in their diagnostic process. Results together showed that there is room to select a best practice from the many practices applied. Such a best practice of PKU-NBS parameters and post-screening parameters could then serve as a generally applicable guideline. Full article

The biomarker-based Dutch Newborn Screening (NBS) panel (as of 2024) comprises 19 inherited metabolic disorders (IMDs). With the use of next-generation sequencing (NGS) as a first-tier screen, NBS could expand to include IMDs that lack a reliable biochemical footprint in dried blood spots, while also reducing secondary findings. To be eligible for inclusion in NBS, an IMD needs to fulfill the Wilson and Jungner criteria, with treatability being one of the most important criteria. In this study, we aimed to identify IMDs eligible for DNA-first NBS when considering only treatability in the context of NBS as a prerequisite. First, three independent reviewers performed a systematic literature review of the 1459 genotypic IMDs and their causative gene(s), as described in the International Classification of Inherited Metabolic Disorders (dated 1 February 2021), applying 16 criteria to exclude non-treatable disorders. Eligible disorders were then discussed in three online meetings with a project group of clinical laboratory geneticists, medical laboratory specialists specialized in IMD, and pediatricians with expertise in IMDs. Based on treatability, we identified 100 genes, causing 95 IMDs, as eligible for NBS, including 42 causal genes for the IMDs in the current biomarker-based NBS. The other 58 genes are primarily associated with treatable defects in amino acid metabolism and fatty acid oxidation. Other IMDs were excluded, most often because of insufficient literature. As the evaluation of treatability was not straightforward, we recommend the development of standardized treatability scores for the inclusion of IMDs in NBS. Full article

In The Netherlands, newborn screening (NBS) for tyrosinemia type 1 (TT1) uses dried blood spot (DBS) succinylacetone (SUAC) as a biomarker. However, high false-positive (FP) rates and a false-negative (FN) case show that the Dutch TT1 NBS protocol is suboptimal. In search of optimization options, we evaluated the protocols used by other NBS programs and their performance. We distributed an online survey to NBS program representatives worldwide (N = 41). Questions focused on the organization and performance of the programs and on changes since implementation. Thirty-three representatives completed the survey. TT1 incidence ranged from 1/13,636 to 1/750,000. Most NBS samples are taken between 36 and 72 h after birth. Most used biomarkers were DBS SUAC (78.9%), DBS Tyrosine (Tyr; 5.3%), or DBS Tyr with second tier SUAC (15.8%). The pooled median cut-off for SUAC was 1.50 µmol/L (range 0.3–7.0 µmol/L). The median cut-off from programs using laboratory-developed tests was significantly higher (2.63 µmol/L) than the medians from programs using commercial kits (range 1.0–1.7 µmol/L). The pooled median cut-off for Tyr was 216 µmol/L (range 120–600 µmol/L). Overall positive predictive values were 27.3% for SUAC, 1.2% for Tyr solely, and 90.1% for Tyr + SUAC. One FN result was reported for TT1 NBS using SUAC, while three FN results were reported for TT1 NBS using Tyr. The NBS programs for TT1 vary worldwide in terms of analytical methods, biochemical markers, and cut-off values. There is room for improvement through method standardization, cut-off adaptation, and integration of new biomarkers. Further enhancement is likely to be achieved by the application of post-analytical tools. Full article

Background: Data suggest that metabolites, other than blood phenylalanine (Phe), better and independently predict clinical outcomes in patients with phenylketonuria (PKU). Methods: To find new biomarkers, we compared the results of untargeted lipidomics and metabolomics in treated adult PKU patients to those of matched controls. Samples (lipidomics in EDTA-plasma (22 PKU and 22 controls) and metabolomics in serum (35 PKU and 20 controls)) were analyzed using ultra-high-performance liquid chromatography and high-resolution mass spectrometry. Data were subjected to multivariate (PCA, OPLS-DA) and univariate (Mann–Whitney U test, p < 0.05) analyses. Results: Levels of 33 (of 20,443) lipid features and 56 (of 5885) metabolite features differed statistically between PKU patients and controls. For lipidomics, findings include higher glycerolipids, glycerophospholipids, and sphingolipids species. Significantly lower values were found for sterols and glycerophospholipids species. Seven features had unknown identities. Total triglyceride content was higher. Higher Phe and Phe catabolites, tryptophan derivatives, pantothenic acid, and dipeptides were observed for metabolomics. Ornithine levels were lower. Twenty-six metabolite features were not annotated. Conclusions: This study provides insight into the metabolic phenotype of PKU patients. Additional studies are required to establish whether the observed changes result from PKU itself, diet, and/or an unknown reason. Full article

In this study, we compare next-generation sequencing (NGS) approaches (targeted panel (tNGS), whole exome sequencing (WES), and whole genome sequencing (WGS)) for application in newborn screening (NBS). DNA was extracted from dried blood spots (DBS) from 50 patients with genetically confirmed inherited metabolic disorders (IMDs) and 50 control samples. One hundred IMD-related genes were analyzed. Two data-filtering strategies were applied: one to detect only (likely) pathogenic ((L)P) variants, and one to detect (L)P variants in combination with variants of unknown significance (VUS). The variants were filtered and interpreted, defining true/false positives (TP/FP) and true/false negatives (TN/FN). The variant filtering strategies were assessed in a background cohort (BC) of 4833 individuals. Reliable results were obtained within 5 days. TP results (47 patient samples) for tNGS, WES, and WGS results were 33, 31, and 30, respectively, using the (L)P filtering, and 40, 40, and 38, respectively, when including VUS. FN results were 11, 13, and 14, respectively, excluding VUS, and 4, 4, and 6, when including VUS. The remaining FN were mainly samples with a homozygous VUS. All controls were TN. Three BC individuals showed a homozygous (L)P variant, all related to a variable, mild phenotype. The use of NGS-based workflows in NBS seems promising, although more knowledge of data handling, automated variant interpretation, and costs is needed before implementation. Full article

Undiagnosed and untreated tyrosinemia type 1 (TT1) individuals carry a significant risk for developing liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Elevated succinylacetone (SA) is pathognomonic for TT1 and therefore often used as marker for TT1 newborn screening (NBS). While SA was long considered to be elevated in every TT1 patient, here we present a recent false-negative SA TT1 screen. A nine-year-old boy presented with HCC in a cirrhotic liver. Additional tests for the underlying cause unexpectedly revealed TT1. Nine years prior, the patient was screened for TT1 via SA NBS with a negative result: SA 1.08 µmol/L, NBS cut-off 1.20 µmol/L. To our knowledge, this report is the first to describe a false-negative result from the TT1 NBS using SA. False-negative TT1 NBS results may be caused by milder TT1 variants with lower SA excretion. Such patients are more likely to be missed in NBS programs and can be asymptomatic for years. Based on our case, we advise TT1 to be considered in patients with otherwise unexplained liver pathology, including fibrosis, cirrhosis and HCC, despite a previous negative TT1 NBS status. Moreover, because the NBS SA concentration of this patient fell below the Dutch cut-off value (1.20 µmol/L at that time), as well as below the range of cut-off values used in other countries (1.29–10 µmol/L), it is likely that false-negative screening results for TT1 may also be occurring internationally. This underscores the need to re-evaluate TT1 SA NBS programs. Full article

The Wilson and Jungner (W&J) and Andermann criteria are meant to help select diseases eligible for population-based screening. With the introduction of next-generation sequencing (NGS) methods for newborn screening (NBS), more inherited metabolic diseases (IMDs) can technically be included, and a revision of the criteria was attempted. This study aimed to formulate statements and investigate whether those statements could elaborate on the criterion of treatability for IMDs to decide on eligibility for NBS. An online Delphi study was started among a panel of Dutch IMD experts (EPs). EPs evaluated, amended, and approved statements on treatability that were subsequently applied to 10 IMDs. After two rounds of Delphi, consensus was reached on 10 statements. Application of these statements selected 5 out of 10 IMDs proposed for this study as eligible for NBS, including 3 IMDs in the current Dutch NBS. The statement: ‘The expected benefit/burden ratio of early treatment is positive and results in a significant health outcome’ contributed most to decision-making. Our Delphi study resulted in 10 statements that can help to decide on eligibility for inclusion in NBS based on treatability, also showing that other criteria could be handled in a comparable way. Validation of the statements is required before these can be applied as guidance to authorities. Full article

Residual heel prick Dried Blood Spots (DBS) are valuable samples for retrospective investigation of inborn metabolic diseases (IMD) and biomarker analyses. Because many metabolites suffer time-dependent decay, we investigated the five-year stability of amino acids (AA) in residual heel prick DBS. In 2019/2020, we analyzed 23 AAs in 2170 residual heel prick DBS from the Dutch neonatal screening program, stored from 2013–2017 (one year at +4 °C and four years at room temperature), using liquid chromatography mass-spectrometry. Stability was assessed by AA changes over the five years. Hydroxyproline could not be measured accurately and was not further assessed. Concentrations of 19 out of the remaining 22 AAs degraded significantly, ranked from most to least stable: aspartate, isoleucine, proline, valine, leucine, tyrosine, alanine, phenylalanine, threonine, citrulline, glutamate, serine, ornithine, glycine, asparagine, lysine, taurine, tryptophan and glutamine. Arginine, histidine and methionine concentrations were below the limit of detection and were likely to have been degraded within the first year of storage. AAs in residual heel prick DBS stored at room temperature are subject to substantial degradation, which may cause incorrect interpretation of test results for retrospective biomarker studies and IMD diagnostics. Therefore, retrospective analysis of heel prick blood should be done in comparison to similarly stored heel prick blood from controls. Full article

In the Netherlands, abnormal New-Born Screening (NBS) results are communicated to parents by the general practitioner (GP). Good communication and consequential trust in professionals is of the utmost importance in the treatment of phenylketonuria (PKU). The aim of this study was to assess parental satisfaction regarding the communication of an abnormal NBS result for PKU in the Netherlands. An email containing the link to a web-based questionnaire was sent by the Dutch PKU Association to their members. Responses to open questions were categorized, data of both open and closed questions were analysed with descriptive statistics and the Chi-Square test using SPSS. Out of 113 parents of a child with PKU (born between 1979 and 2020), 68 stated they were overall unsatisfied with the first communication of the NBS result. Seventy-five parents indicated that wrong or no information about PKU was given. A significant decrease was found in the number of parents being contact by their own GP over the course of 40 years (p < 0.05). More than half of all parents were overall unsatisfied with the first communication of the abnormal NBS result for PKU. Further research on how to optimize communication of an abnormal NBS results is necessary. Full article

Purpose: this systematic review aimed to assess the effects of dietary liberalization following tetrahydrobiopterin (BH₄) treatment on anthropometric measurements, nutritional biomarkers, quality of life, bone density, mental health and psychosocial functioning, and burden of care in PKU patients. Methods: the PubMed, Cochrane, and Embase databases were searched on 7 April 2022. We included studies that reported on the aforementioned domains before and after dietary liberalization as a result of BH₄ treatment in PKU patients. Exclusion criteria were: studies written in a language other than English; studies that only included data of a BH₄ loading test; insufficient data for the parameters of interest; and wrong publication type. Both within-subject and between-subject analyses were assessed, and meta-analyses were performed if possible. Results: twelve studies containing 14 cohorts and 228 patients were included. Single studies reported few significant differences. Two out of fifteen primary meta-analyses were significant; BMI was higher in BH₄-treated patients versus controls (p = 0.02; standardized mean difference (SMD) (95% confidence interval (CI)) = −0.37 (−0.67, −0.06)), and blood cholesterol concentrations increased after starting BH₄ treatment (p = 0.01; SMD (CI) = −0.70 (−1.26, −0.15)). Conclusion: there is no clear evidence that dietary liberalization after BH₄ treatment has a positive effect on anthropometric measurements, nutritional biomarkers, or quality of life. No studies could be included for bone density, mental health and psychosocial functioning, and burden of care. Full article

Newborn screening (NBS) aims to identify neonates with severe conditions for whom immediate treatment is required. Currently, a biochemistry-first approach is used to identify these disorders, which are predominantly inherited meta1bolic disorders (IMD). Next-generation sequencing (NGS) is expected to have some advantages over the current approach, for example the ability to detect IMDs that meet all screening criteria but lack an identifiable biochemical footprint. We have now designed a technical study to explore the use of NGS techniques as a first-tier approach in NBS. Here, we describe the aim and set-up of the NGS-first for the NBS (NGSf4NBS) project, which will proceed in three steps. In Step 1, we will identify IMDs eligible for NGS-first testing, based on treatability. In Step 2, we will investigate the feasibility, limitations and comparability of different technical NGS approaches and analysis workflows for NBS, eventually aiming to develop a rapid NGS-based workflow. Finally, in Step 3, we will prepare for the incorporation of this workflow into the existing Dutch NBS program and propose a protocol for referral of a child after a positive NGS test result. The results of this study will be the basis for an additional analytical route within NBS that will be further studied for its applicability within the NBS program, e.g., regarding the ethical, legal, financial and social implications. Full article

Phenylketonuria and tyrosinemia type 1 are treated with dietary phenylalanine (Phe) restriction. Aspartame is a Phe-containing synthetic sweetener used in many products, including many ‘regular’ soft drinks. Its amount is (often) not declared; therefore, patients are advised not to consume aspartame-containing foods. This study aimed to determine the variation in aspartame concentrations and its Phe-containing degradation products in aspartame-containing soft drinks. For this, an LC–MS/MS method was developed for the analysis of aspartame, Phe, aspartylphenylalanine, and diketopiperazine in soft drinks. In total, 111 regularly used soft drinks from 10 European countries were analyzed. The method proved linear and had an inter-assay precision (CV%) below 5% for aspartame and higher CVs% of 4.4–49.6% for the degradation products, as many concentrations were at the limit of quantification. Aspartame and total Phe concentrations in the aspartame-containing soft drinks varied from 103 to 1790 µmol/L (30–527 mg/L) and from 119 to 2013 µmol/L (20–332 mg/L), respectively, and were highly variable among similar soft drinks bought in different countries. Since Phe concentrations between drinks and countries highly vary, we strongly advocate the declaration of the amount of aspartame on soft drink labels, as some drinks may be suitable for consumption by patients with Phe-restricted diets. Full article

Tyrosinemia type 1 (TT1) treatment with 2-(2-nitro-4-trifluormethyl-benzyl)-1,3-cyclohexanedione (NTBC) and a phenylalanine-tyrosine restricted diet is associated with low phenylalanine concentrations. Phenylalanine supplementation is prescribed without comprehensive consideration about its effect on metabolic control. We investigated the effect of phenylalanine supplementation on bloodspot phenylalanine, tyrosine, NTBC and succinylacetone. Eleven TT1 patients received 0, 20 and 40 mg/kg/day phenylalanine supplementation with the phenylalanine-tyrosine free L-amino acid supplements. Bloodspots were collected before breakfast, midday and evening meal. Differences between study periods, sample times and days within a study period were studied using (generalized) linear mixed model analyses. Twenty and 40 mg/kg/day phenylalanine supplementation prevented daytime phenylalanine decreases (p = 0.05) and most low phenylalanine concentrations, while tyrosine concentrations increased (p < 0.001). Furthermore, NTBC and succinylacetone concentrations did not differ between study periods. To conclude, 20 mg/kg/day phenylalanine supplementation can prevent most low phenylalanine concentrations without increasing tyrosine to concentrations above the target range or influencing NTBC and succinylacetone concentrations, while 40 mg/kg/day increased tyrosine concentrations to values above the targeted range. Additionally, this study showed that the effect of phenylalanine supplementation, and a possible phenylalanine deficiency, should be assessed using pre-midday meal blood samples that could be combined with an overnight fasted sample when in doubt. Full article