International Journal of Neonatal Screening

8 pages, 606 KB

Open AccessCase Report

Mitochondrial Acetoacetyl-CoA Thiolase Deficiency: Three New Cases Detected by Newborn Screening Confirming the Significance of C4OH Elevation

by Alessandra Vasco, Clarissa Berardo, Simona Lucchi, Laura Cappelletti, Giulio Tamburello, Salvatore Fazzone, Alessia Mauri, Francesca Fiumani, Diana Postorivo, Luisella Alberti, Michela Perrone Donnorso, Serena Gasperini, Francesca Furlan, Laura Fiori, Stephana Carelli, Laura Assunta Saielli, Cristina Montrasio and Cristina Cereda

Int. J. Neonatal Screen. 2025, 11(3), 76; https://doi.org/10.3390/ijns11030076 - 6 Sep 2025

Abstract

Acetoacetyl-CoA thiolase deficiency, also known as Beta-ketothiolase deficiency (BKTD), is an autosomal recessive organic aciduria included in the Italian newborn screening (NBS) panel. It is caused by mutations in the ACAT1 gene, which encodes the mitochondrial acetyl-CoA acetyltransferase. Its deficiency impairs the degradation [...] Read more.

Acetoacetyl-CoA thiolase deficiency, also known as Beta-ketothiolase deficiency (BKTD), is an autosomal recessive organic aciduria included in the Italian newborn screening (NBS) panel. It is caused by mutations in the ACAT1 gene, which encodes the mitochondrial acetyl-CoA acetyltransferase. Its deficiency impairs the degradation of isoleucine and acetoacetyl-CoA, leading to the accumulation of toxic metabolites. We describe three cases of BKTD. The first newborn showed increase in C5:1, C4DC/C5OH, C3DC/C4OH in the NBS. Urinary organic acids (uOAs) revealed marked excretion of 2-methyl-3-hydroxybutyrate. Tiglylglycine was absent. Genetic testing identified the compound heterozygosity for two pathogenic ACAT1 variants. The second patient showed increased levels of C5:1, C4DC/C5OH, C3DC/C4OH in the NBS. uOAs revealed 2-methyl-3-hydroxybutyrate and tiglylglycine. A homozygous VUS in ACAT1 was identified. The third case showed elevation of C4DC/C5OH, C3DC/C4OH in the NBS, with a slight increase in C5:1. uOAs showed 2-methyl-3-hydroxybutyrate and tiglylglycine. A homozygous missense VUS was identified in the ACAT1 gene. BKTD exhibited variable NBS biochemical phenotypes across the three cases. While C5OH and C5:1, the primary markers, were not consistently elevated in all our cases, C4OH strongly increased in all three. Our findings support the use of C4OH in a combined marker strategy to improve BKTD NBS. Full article

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16 pages, 2770 KB

Open AccessArticle

Comparing DNA Isolation and Preparation Protocols for Dried Blood Spots in the Context of Genomic Newborn Screening

by Annelotte J. Duintjer, Sandra Imholz, Ingrid Pico-Knijnenburg, Adinda Heuperman, Hennie Hodemaekers, Eva S. Deutekom, Els Voorhoeve, Martijn E. T. Dollé and Mirjam van der Burg

Int. J. Neonatal Screen. 2025, 11(3), 75; https://doi.org/10.3390/ijns11030075 - 3 Sep 2025

Abstract

Due to rapid technical advancements and increasing cost-effectiveness, the potential application of next-generation sequencing (NGS) in newborn screening (NBS) has raised great interest worldwide. Genomic NBS offers the possibility to improve current NBS programs when applied as follow-up tier, and, as first-tier, allows [...] Read more.

Due to rapid technical advancements and increasing cost-effectiveness, the potential application of next-generation sequencing (NGS) in newborn screening (NBS) has raised great interest worldwide. Genomic NBS offers the possibility to improve current NBS programs when applied as follow-up tier, and, as first-tier, allows for inclusion of conditions lacking a detectable biomarker for conventional NBS. Obtaining enough high-quality DNA from typically limited dried blood spot (DBS) material to meet NGS requirements can be challenging. Selecting a DNA isolation method for genomic NBS requires balancing technical performance and laboratory feasibility with optimal cost-effectiveness. Ten DNA isolation protocols, including two column-based, five lysis-based, and three semi-automated magnetic bead-based protocols, were evaluated on technical outcomes and performance in targeted amplicon sequencing. Additionally, estimated costs, hands-on time, turnaround time, scalability, and plastic footprint were assessed. Although technical outcomes, including yield, purity, and molecular weight, differed between methods, qualitative results in amplicon sequencing, as defined by read output, mapping, and coverage depth, were found sufficient and comparable for various protocols. In conclusion, both technical requirements and operational parameters are crucial when selecting a DNA isolation protocol and will depend on the NGS application as well as the NBS approach, as either first-tier or follow-up tier. Full article

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16 pages, 1147 KB

Open AccessArticle

Umbilical Cord Blood Sampling for Newborn Screening of Pompe Disease and the Detection of a Novel Pathogenic Variant and Pseudodeficiency Variants in an Asian Population

by Fook-Choe Cheah, Sharifah Azween Syed Omar, Jasmine Lee, Zheng Jiet Ang, Anu Ratha Gopal, Wan Nurulhuda Wan Md Zin, Beng Kwang Ng, Shu-Chuan Chiang and Yin-Hsiu Chien

Int. J. Neonatal Screen. 2025, 11(3), 74; https://doi.org/10.3390/ijns11030074 - 3 Sep 2025

Abstract

Pompe disease is an autosomal recessive metabolic disorder caused by acid alpha-glucosidase (GAA) deficiency. The use of umbilical cord blood (UCB) for newborn screening (NBS) of Pompe disease, compared to heel-prick sampling, has not been widely studied. This study compared GAA activity in [...] Read more.

Pompe disease is an autosomal recessive metabolic disorder caused by acid alpha-glucosidase (GAA) deficiency. The use of umbilical cord blood (UCB) for newborn screening (NBS) of Pompe disease, compared to heel-prick sampling, has not been widely studied. This study compared GAA activity in UCB from term newborns with peripheral or heel-prick blood samples obtained on days 1, 2, and 3 after birth. Enzyme assays were performed using UPLC-MS/MS. Sanger sequencing was conducted in infants with low GAA activity to identify pathogenic variants. Among 4091 UCB samples analyzed over 18 months, the mean GAA activity was 10.04 ± 5.95 μM/h, higher in females than males [Median (IQR): 9.83 (5.45) vs. 9.08 (4.97) μM/h, respectively, p < 0.001], and similar across ethnicities. GAA levels in UCB and Day 3 heel-prick samples were comparable. A GAA cut-off value of 1.54 μM/h (0.1% of study population) identified one infant (0.024% prevalence) with a novel bi-allelic variant—c.2005_2010del (p.Pro669_Phe670del) and c.1123C>T (p.Arg375Cys), and 12 infants with non-pathogenic pseudodeficiency alleles. This study supports GAA measurement in UCB as a viable alternative for NBS, with enzyme activity remaining stable for up to 72 h post-collection. Larger-scale multicenter nationwide studies are warranted to confirm this prevalence in our population. Full article

(This article belongs to the Special Issue Advances in Newborn Screening for Lysosomal Disorders: From Laboratory Screening to Diagnosis)

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11 pages, 216 KB

Open AccessArticle

Next-Generation Sequencing in the Diagnostic Workup of Neonatal Dried Blood Spot Screening in Sweden 2015–2023

by Lene Sörensen, Jorge Asin-Cayuela, Michela Barbaro, Helene Bruhn, Martin Engvall, Nicole Lesko, Karin Naess, Mikael Oscarson, Yan Shen, Malin Ueberschär, Anna Wredenberg, Fredrik H. Sterky, Anna Wedell and Rolf H. Zetterström

Int. J. Neonatal Screen. 2025, 11(3), 73; https://doi.org/10.3390/ijns11030073 - 3 Sep 2025

Abstract

Sweden has one neonatal screening laboratory and two centers conducting diagnostic workup for inborn errors of metabolism (IEM). Next-generation sequencing (NGS) has been gradually introduced as a confirmatory diagnostic test in the Swedish newborn screening program. Here, we describe the use of NGS [...] Read more.

Sweden has one neonatal screening laboratory and two centers conducting diagnostic workup for inborn errors of metabolism (IEM). Next-generation sequencing (NGS) has been gradually introduced as a confirmatory diagnostic test in the Swedish newborn screening program. Here, we describe the use of NGS in the diagnostic workup of IEM in screening-detected babies in Sweden between 2015 and 2023. During this period, 1,023,344 newborn children were screened, and 81 of 290 IEM cases were genetically confirmed using NGS. Planned improvements to the program are to perform genetic validation directly on the initial dried blood spot (DBS). As whole-genome sequencing (WGS) is superior in detecting causative genetic variants compared to Sanger sequencing, targeted NGS, and whole-exome sequencing (WES), it will likely become the method of choice more broadly in the future. A strong focus is to consolidate the nationally coordinated DBS newborn screening program, with all its individual components, including screening, targeted diagnostics, individualized treatment, and follow-up. This challenges the current regionalized organization of Swedish healthcare, which hinders close national collaboration between experts and sharing of data, as well as equal access to advanced treatments for identified patients, regardless of their place of birth. Full article

5 pages, 983 KB

Open AccessCorrection

Correction: Kuypers et al. Evaluation of Neonatal Screening Programs for Tyrosinemia Type 1 Worldwide. Int. J. Neonatal Screen. 2024, 10, 82

by Allysa M. Kuypers, Marelle J. Bouva, J. Gerard Loeber, Anita Boelen, Eugenie Dekkers, Konstantinos Petritis, C. Austin Pickens, The ISNS Representatives, Francjan J. van Spronsen and M. Rebecca Heiner-Fokkema

Int. J. Neonatal Screen. 2025, 11(3), 72; https://doi.org/10.3390/ijns11030072 - 1 Sep 2025

Abstract

The authors wish to make the following correction to their paper published in the International Journal of Neonatal Screening [...] Full article

14 pages, 1122 KB

Open AccessArticle

Optimization of the Performance of Newborn Screening for X-Linked Adrenoleukodystrophy by Flow Injection Analysis Tandem Mass Spectrometry

by Chengfang Tang, Minyi Tan, Yanna Cai, Sichi Liu, Ting Xie, Xiang Jiang, Li Tao, Yonglan Huang and Fang Tang

Int. J. Neonatal Screen. 2025, 11(3), 71; https://doi.org/10.3390/ijns11030071 - 29 Aug 2025

Abstract

The aim of this study was to improve screening efficiency by establishing reasonable interpretation criteria for the use of flow injection analysis tandem mass spectrometry (FIA-MS/MS) in newborn screening (NBS) for X-linked adrenoleukodystrophy (X-ALD). FIA-MS/MS was employed to analyze very-long-chain acylcarnitines (ACs) and [...] Read more.

The aim of this study was to improve screening efficiency by establishing reasonable interpretation criteria for the use of flow injection analysis tandem mass spectrometry (FIA-MS/MS) in newborn screening (NBS) for X-linked adrenoleukodystrophy (X-ALD). FIA-MS/MS was employed to analyze very-long-chain acylcarnitines (ACs) and lysophosphatidylcholines (LPCs) and their ratios in dried blood spot (DBS) obtained from five X-ALD patients in the neonatal period (0–7 days old) and 7123 healthy neonate controls. By comparing these results and analyzing receiver operating characteristic (ROC) curves, we identified sensitive indicators for X-ALD screening in newborns. To evaluate the performance of different FIA-MS/MS screening indicators, we simultaneously analyzed 7712 neonatal DBS samples obtained for X-ALD screening using FIA-MS/MS and the established liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantitative detection of C26:0-lysophosphatidylcholine (C26:0-LPC). Furthermore, 84,268 newborn X-ALD screening results were retrospectively analyzed to further evaluate the screening performance of FIA-MS/MS. After the three-step optimization evaluation, the optimized first-tier sensitive screening indicators of FIA-MS/MS were C24:0-AC, C26:0LPC, and C24:0/C22:0-AC. Among the 7712 newborns screened, one case was confirmed to be double-positive. Within separate statistical analyses, based on LC-MS/MS screening alone (positive cutoff > 0.17 µmol/L), only seven cases (0.09%) were initially positive, with a positive predictive value (PPV) of 42.8%, and two additional ABCD1 VUS hemizygous males were detected. Through the retrospective analysis of 84,268 newborns, eight ABCD1 variants (six hemizygous males and two heterozygous females) were ultimately identified. Our study showed that the optimization of first-tier screening performance is particularly important if second-tier screening is not performed. Using LC-MS/MS for second-tier screening for X-ALD can significantly reduce the number of false positives, but the method still misses some false negatives. If it is used as a first-tier assessment, more VUS variant neonates can be detected. Full article

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14 pages, 6618 KB

Open AccessArticle

Learning Collaborative to Support Continuous Quality Improvement in Newborn Screening

by Elizabeth Jones, Sikha Singh, Sarah McKasson, Ruthanne Sheller, Jelili Ojodu and Ashley Comer

Int. J. Neonatal Screen. 2025, 11(3), 70; https://doi.org/10.3390/ijns11030070 - 27 Aug 2025

Abstract

As newborn screening (NBS) programs deal with growing complexities, including adding new disorders to their screening panels, adopting new technologies/screening methods, and workforce shortages, there is a greater need for continuous quality improvement (CQI) to ensure the NBS system is meeting its primary [...] Read more.

As newborn screening (NBS) programs deal with growing complexities, including adding new disorders to their screening panels, adopting new technologies/screening methods, and workforce shortages, there is a greater need for continuous quality improvement (CQI) to ensure the NBS system is meeting its primary goal of identifying infants with NBS disorders in a timely fashion. In 2019, the Health Resources and Services Administration’s (HRSA) Maternal and Child Health Bureau (MCHB) awarded funding to the Association of Public Health Laboratories’ (APHL) Newborn Screening Technical assistance and Evaluation Program (NewSTEPs) to address CQI in the NBS system through a collaborative, data-driven process. From 2019–2024, NewSTEPs funded 36 quality improvement (QI) projects from a variety of state NBS programs and research centers across the U.S., to address timeliness, detection of out-of-range results, communication of results, and/or confirmation of diagnosis. Thirty-three QI teams completed their projects, and 85% achieved their specified goal outlined in their aim statement. Despite limitations, the QI Projects Collaborative provided NBS programs with funding and resources to begin and sustain quality improvement initiatives. This model of a technical assistance and central resource center for CQI was effective in achieving quality improvements within the national NBS system. Full article

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6 pages, 220 KB

Open AccessArticle

Evaluating the Impact of Newborn Screening for Cystic Fibrosis in Portugal: A Decade of Insights and Outcomes

by Bernardo Camacho, Luísa Pereira, Raquel Bragança, Susana Castanhinha, Raquel Penteado, Teresa R. Silva, Pedro Miragaia, Sónia Silva, Ana L. Cardoso, Telma Barbosa, Cristina Freitas, Juan Gonçalves, Ana Marcão, Laura Vilarinho, Celeste Barreto and Carolina Constant

Int. J. Neonatal Screen. 2025, 11(3), 69; https://doi.org/10.3390/ijns11030069 - 27 Aug 2025

Abstract

The implementation of newborn screening (NBS) has revolutionized the diagnostic landscape of cystic fibrosis (CF). In Portugal, NBS was initiated in October 2013 through a pilot study and was subsequently fully integrated into a nationwide program by December 2018. Infants with positive screening [...] Read more.

The implementation of newborn screening (NBS) has revolutionized the diagnostic landscape of cystic fibrosis (CF). In Portugal, NBS was initiated in October 2013 through a pilot study and was subsequently fully integrated into a nationwide program by December 2018. Infants with positive screening results are referred to a specialized CF reference center for diagnostic confirmation, employing Sweat Chloride Testing (SCT) and genetic testing for CFTR variants. We aimed to analyze infants with a positive CF screening and determine the false positive and false negative rates, as well as to calculate the positive predictive value and sensitivity of our NBS program. A retrospective nationwide analysis was conducted on infants with a positive NBS for CF between October 2013 and February 2023. Two hundred and forty infants were referred from the NBS program; 74 (30.8%) were confirmed to have CF through SCT and genetic testing. Sensitivity was 93.2%, and the positive predictive value (PPV) was 30.8%. In addition, 48.5% were homozygous for F508del variants, and 87.8% had at least one F508del variant. Guidelines set forth by the European Cystic Fibrosis Society advise NBS programs to achieve a minimum PPV of 30% and a minimum sensitivity of 95%. Our report demonstrated good compliance with these recommendations. Full article

(This article belongs to the Special Issue Advances in Cystic Fibrosis Newborn Screening: From Laboratory Testing to Diagnosis)

9 pages, 250 KB

Open AccessArticle

Novel Phenotypic Insights into the IDS c.817C>T Variant in Mucopolysaccharidosis Type II from Newborn Screening Cohorts

by Éliane Beauregard-Lacroix, Caitlin Menello, Madeline Steffensen, Hsiang-Yu Lin, Chih-Kuang Chuang, Shuan-Pei Lin and Can Ficicioglu

Int. J. Neonatal Screen. 2025, 11(3), 68; https://doi.org/10.3390/ijns11030068 - 26 Aug 2025

Abstract

Mucopolysaccharidosis (MPS) type II, or Hunter syndrome, is an X-linked lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase. Glycosaminoglycan (GAG) accumulation leads to progressive multisystemic involvement, with coarse facial features, hepatosplenomegaly, short stature, recurrent upper respiratory infections, hearing loss, hernias, dysostosis multiplex, [...] Read more.

Mucopolysaccharidosis (MPS) type II, or Hunter syndrome, is an X-linked lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase. Glycosaminoglycan (GAG) accumulation leads to progressive multisystemic involvement, with coarse facial features, hepatosplenomegaly, short stature, recurrent upper respiratory infections, hearing loss, hernias, dysostosis multiplex, joint contractures, and cardiac valve disease. Individuals with the neuronopathic form of the disease also have central nervous system (CNS) involvement with developmental delay and progressive cognitive decline. Enzyme replacement therapy (ERT), idursulfase, is the only FDA-approved treatment for MPS II. MPS II was added to the Recommended Uniform Screening Panel (RUSP) in the United States in 2022, and screening is ongoing in several other countries, including Taiwan. Here, we report seven individuals from four families identified through newborn screening sharing the same IDS variant: c.817C>T, p.Arg273Trp. Confirmatory testing demonstrated low iduronate-2-sulfatase activity level and elevated GAGs in every individual, but they had no signs or symptoms of MPS II. They were aged 8 months to 60 years old according to the most recent assessment and all remained asymptomatic. ERT was not initiated for any of them. Our findings suggest that the IDS c.817C>T variant is associated with abnormal biochemical findings but no clinical phenotype of MPS II. Newborn screening will likely identify additional cases and provide a better understanding of the clinical significance of this variant. Full article

(This article belongs to the Special Issue Advances in Newborn Screening for Lysosomal Disorders: From Laboratory Screening to Diagnosis)

12 pages, 776 KB

Open AccessArticle

Exceptionally High Cystic Fibrosis-Related Morbidity and Mortality in Infants and Young Children in India: The Need for Newborn Screening and CF-Specific Capacity Building

by Priyanka Medhi, Grace R. Paul, Madhan Kumar, Grace Rebekah, Philip M. Farrell, Jolly Chandran, Rekha Aaron, Aaron Chapla and Sneha D. Varkki

Int. J. Neonatal Screen. 2025, 11(3), 67; https://doi.org/10.3390/ijns11030067 - 22 Aug 2025

Abstract

Early diagnosis of cystic fibrosis (CF) through newborn screening (NBS) improves clinical outcomes, but in countries like India, delayed diagnosis increases morbidity, mortality, and likely underestimates infant deaths from CF. We performed a retrospective study at a single center in south India from [...] Read more.

Early diagnosis of cystic fibrosis (CF) through newborn screening (NBS) improves clinical outcomes, but in countries like India, delayed diagnosis increases morbidity, mortality, and likely underestimates infant deaths from CF. We performed a retrospective study at a single center in south India from 2017 to 2025 reviewing children diagnosed with CF before one year of age. Patient demographic, clinical, and genetic data were analyzed to characterize early clinical features and identify factors linked to mortality. Of 56 infants diagnosed with CF, 59% survived (median current age 55 months) while 41% died (median age of death 5 months). Key clinical indicators included sibling death with CF-like symptoms, rapid weight loss, and persistent respiratory or nutritional complications. Mortality risk under one year was significantly linked to hypoalbuminemia (OR 9.7), severe malnutrition (OR 4.4), severe anemia (hemoglobin < 7 g/dL) requiring blood transfusions (OR 3.0), and peripheral edema (OR 4.2). A triad of anemia, hypoalbuminemia, and edema was found to strongly predict death (OR 4.2). Integrating clinical checklists of these manifestations into primary healthcare may improve prompt referrals for earlier diagnosis and treatment. Continued education and advocacy for NBS are essential to reduce potentially preventable CF-related deaths in young children. Full article

(This article belongs to the Special Issue Advances in Cystic Fibrosis Newborn Screening: From Laboratory Testing to Diagnosis)

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8 pages, 279 KB

Open AccessCase Report

MCT8 Deficiency in Infancy: Opportunities for Early Diagnosis and Screening

by Ilja Dubinski, Belana Debor, Sofia Petrova, Katharina A. Schiergens, Heike Weigand and Heinrich Schmidt

Int. J. Neonatal Screen. 2025, 11(3), 66; https://doi.org/10.3390/ijns11030066 - 21 Aug 2025

Abstract

Background: Monocarboxylate-transporter-8-(MCT8) deficiency, or Allan–Herndon–Dudley syndrome (AHDS), is a rare X-linked disorder caused by pathogenic variants in the SLC16A2 gene, leading to impaired transport of thyroid hormones, primarily T3 and T4, across cell membranes. The resulting central hypothyroidism and peripheral hyperthyroidism cause neurodevelopmental [...] Read more.

Background: Monocarboxylate-transporter-8-(MCT8) deficiency, or Allan–Herndon–Dudley syndrome (AHDS), is a rare X-linked disorder caused by pathogenic variants in the SLC16A2 gene, leading to impaired transport of thyroid hormones, primarily T3 and T4, across cell membranes. The resulting central hypothyroidism and peripheral hyperthyroidism cause neurodevelopmental impairment and thyrotoxicosis. Despite the availability of therapy options, e.g., with triiodothyroacetic acid (TRIAC), diagnosis is often delayed, partly due to normal TSH levels or incomplete genetic panels. MCT8 deficiency is not yet included in newborn-screening programs worldwide. Case Description: We present a case of an infant genetically diagnosed with MCT8 deficiency at 5 months of age after presenting with muscular hypotonia, lack of head control, and developmental delay. Thyroid function testing revealed a normal TSH, low free T4, and significantly elevated free T3 and free T3/T4 ratio. Treatment with TRIAC (Emcitate^®) was initiated promptly, with close drug monitoring. Despite persistent motor deficits and dystonia, some developmental progress was observed, as well as reduction in hyperthyroidism. Discussion/Conclusions: This case underscores the importance of early free T3 and fT3/fT4 ratio testing in infants with unexplained developmental delay. Broader inclusion of SLC16A2 in genetic panels and consideration of newborn screening could improve early diagnosis and outcomes in this rare but treatable condition. Full article

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12 pages, 268 KB

Open AccessReview

Is It Time to Expand Newborn Screening for Congenital Hypothyroidism to Other Rare Thyroid Diseases?

by Antonella Olivieri, Maria Cristina Vigone, Mariacarolina Salerno and Luca Persani

Int. J. Neonatal Screen. 2025, 11(3), 65; https://doi.org/10.3390/ijns11030065 - 20 Aug 2025

Abstract

Congenital hypothyroidism (CH) is a heterogeneous condition present at birth, resulting in severe-to-mild thyroid hormone deficiency. This condition is difficult to recognize shortly after birth. Therefore, many countries worldwide have implemented newborn screening (NBS) programs for CH since the 1970s. The most recent [...] Read more.

Congenital hypothyroidism (CH) is a heterogeneous condition present at birth, resulting in severe-to-mild thyroid hormone deficiency. This condition is difficult to recognize shortly after birth. Therefore, many countries worldwide have implemented newborn screening (NBS) programs for CH since the 1970s. The most recent European guidelines strongly recommend screening for primary CH, as well as for central CH when financial resources are available. However, no consensus has been reached yet to screen more rare forms of CH, such as Allan–Herndon–Dudley syndrome (AHDS), an X-linked condition linked to mutations in the gene encoding a transmembrane monocarboxylate transporter (MCT8), resistance to thyroid hormone beta (RTHβ), and resistance to thyroid hormone alfa (RTHα). The combined measurement of thyroid-stimulating hormone (TSH) and total thyroxine (TT4) on DBS currently allows the recognition of central CH (TSH low/normal and low TT4 without defects in transport proteins). With the introduction of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for measurement of free triiodothyronine (FT3) and free thyroxine (FT4), it would be possible to screen for RTHβ (TSH normal/high and high FT4). More complicated would be the method to screen RTHα. It would require the combined measurement of FT4 and FT3 and the determination of FT3/FT4 ratio, while the combined measurement of FT3 and reverse T3 (rT3) to calculate FT3/rT3 ratio would be useful to screen AHDS. In this article, we provide some reflections on expanding NBS for primary CH also to other rare forms of CH. Full article

(This article belongs to the Special Issue Newborn Screening for Congenital Hypothyroidism)

15 pages, 4045 KB

Open AccessArticle

Newborn Screening Program for Spinal Muscular Atrophy in the Campania Region (Italy): Current Limitations and Potential Perspectives

by Adelaide Ambrosio, Tiziana Fioretti, Barbara D’Andrea, Lucia Pezone, Ilaria Bitetti, Carmela Di Domenico, Sabrina Vallone, Valeria Maiolo, Angela Cioce, Mariano Giustino, Antonio Varone and Gabriella Esposito

Int. J. Neonatal Screen. 2025, 11(3), 64; https://doi.org/10.3390/ijns11030064 - 17 Aug 2025

Abstract

Three targeted therapies are currently available for spinal muscular atrophy (SMA), which have dramatically changed the natural history of this severe and potentially fatal disease. More than 95% of SMA cases have a homozygous deletion of exon 7 of the SMN1 gene. Disease [...] Read more.

Three targeted therapies are currently available for spinal muscular atrophy (SMA), which have dramatically changed the natural history of this severe and potentially fatal disease. More than 95% of SMA cases have a homozygous deletion of exon 7 of the SMN1 gene. Disease expression mainly depends on the copy number of SMN2, a hypomorphic copy of SMN1. Many countries in the world have implemented newborn screening (NBS) programs for early identification and treatment of children with SMA. We herein present the first two-year results of the SMA NBS program in Campania, a region with one of the highest birth rates in Italy. Genomic DNA was extracted from dried blood spots (DBS) and peripheral blood. For DBS, the SMN1 gene copy number was evaluated by quantitative polymerase chain reaction (qPCR) targeting SMN1 exon 7 and a reference gene (RPP30). In positive newborns and their parents, SMN1/SMN2 copies were evaluated by multiplex ligation probe amplification (MLPA). We analyzed 77,945 newborns and identified 11 positive children. Six patients had 2 copies of SMN2, but only one showed severe SMA-related signs at birth. Eligible newborns were treated with gene therapy within 20 days of birth. Notably, qPCR failed to amplify the reference RPP30 gene in 10/77,945 DBS. Despite this limitation, we observed that about 1/40 DBS had ΔCt values consistent with the presence of one SMN1 copy. The semi-automated procedure used for SMA NBS showed excellent performance in detecting the presence of homozygous deletion of SMN1 exon 7, with the exception of a few cases with the absence of amplification of the reference gene. By solving this limitation, the screening procedure has the potential to detect heterozygous carriers of the SMN1 deletion and, consequently, identify families at procreative risk of SMA. Full article

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12 pages, 1071 KB

Open AccessArticle

Seasonal Fluctuations and Stability of Adenosine in Dried Blood Spots for Neonatal Screening

by Xiangchun Yang, Jing Liu, Xia Li, Dongyang Hong, Shanshan Wu, Changshui Chen and Haibo Li

Int. J. Neonatal Screen. 2025, 11(3), 63; https://doi.org/10.3390/ijns11030063 - 13 Aug 2025

Abstract

Seasonal and environmental factors, including temperature, humidity, and storage conditions, significantly impact the stability of biochemical markers in dried blood spot (DBS) samples. This study investigates these influences specifically for adenosine (ADO) levels, a critical biomarker for neonatal screening of adenosine deaminase (ADA) [...] Read more.

Seasonal and environmental factors, including temperature, humidity, and storage conditions, significantly impact the stability of biochemical markers in dried blood spot (DBS) samples. This study investigates these influences specifically for adenosine (ADO) levels, a critical biomarker for neonatal screening of adenosine deaminase (ADA) deficiency. This study analyzed seasonal fluctuations in ADO concentrations across three regions in China (Ningbo, Nanjing, and Changsha) over 11 months, and evaluated ADO stability under different storage conditions (4 °C, 20 °C, and 40 °C). ADO levels demonstrated significant seasonal variability, peaking in July–August. Median concentrations increased by 111–189% in warmer months compared to winter across all sites. Storage experiments showed that ADO was most stable at 4 °C (fluctuations < 5% over 7 days), while levels at 40 °C increased by 18%. Re-adjusting the ADO reference range based on seasonal data reduced false positive rates from 2.48% to 0.15%, a 94% reduction. This study underscores the necessity of implementing seasonally dynamic reference ranges and strict cold-chain storage (4 °C) to enhance screening accuracy for ADA deficiency. The findings provide a robust foundation for optimizing neonatal screening protocols globally, especially in regions with distinct seasonal climates. Full article

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12 pages, 234 KB

Open AccessArticle

Communication of an Abnormal Metabolic Newborn Screening Result in the Netherlands: A Qualitative Exploratory Study of the General Practitioner’s Perspective

by Sietske Haitjema, Charlotte M. A. Lubout, Justine H. M. Zijlstra, Rendelien K. Verschoof-Puite and Francjan J. van Spronsen

Int. J. Neonatal Screen. 2025, 11(3), 62; https://doi.org/10.3390/ijns11030062 - 8 Aug 2025

Abstract

Newborn screening (NBS) for inherited metabolic diseases (IMD) aims to find children in which immediate action can prevent severe symptoms. We previously studied parental satisfaction with the communication of the NBS result for phenylketonuria, which in the Netherlands is done by the general [...] Read more.

Newborn screening (NBS) for inherited metabolic diseases (IMD) aims to find children in which immediate action can prevent severe symptoms. We previously studied parental satisfaction with the communication of the NBS result for phenylketonuria, which in the Netherlands is done by the general practitioners (GPs). More than half of all parents were unsatisfied with the communication of the abnormal NBS result. The aim of this qualitative exploratory study was to portray a number of GPs’ opinions and experiences in communicating an abnormal metabolic NBS result. We performed semi-structured interviews with ten GPs to evaluate the process of communicating the abnormal NBS result. An additional two GPs provided their answers via email. The data revealed four key themes: (1) dealing with the urgency of the metabolic NBS result, (2) the role of the GP in the NBS process, (3) the current organization of NBS in the Netherlands and (4) evaluating roles and responsibilities in communicating abnormal metabolic NBS results. Despite the willingness of GPs to inform parents about NBS results, it is questionable whether they have the necessary tools to effectively conduct these conversations given their limited experience with IMDs. In light of the increasing number of diseases in the NBS program, it would be interesting to explore alternative tools for communicating the NBS result to parents. Full article

11 pages, 746 KB

Open AccessArticle

Prediction of Congenital Portosystemic Shunt in Neonatal Hypergalactosemia Using Gal-1-P/Gal Ratio, Bile Acid, and Ammonia

by Sayaka Suzuki-Ajihara, Ikuma Musha, Masato Arao, Koki Mori, Shunsuke Fujibayashi, Ihiro Ryo, Tomotaka Kono, Asako Tajima, Hiroshi Mochizuki, Atsuko Imai-Okazaki, Ryuichiro Araki, Chikahiko Numakura and Akira Ohtake

Int. J. Neonatal Screen. 2025, 11(3), 61; https://doi.org/10.3390/ijns11030061 - 7 Aug 2025

Abstract

Congenital portosystemic shunts (CPSSs) are often associated with life-threatening systemic complications, which may be detected by identifying hypergalactosemia in newborn screening (NBS). However, diagnosing CPSS at an early stage is not easy. The purpose of this study was to predict CPSS early using [...] Read more.

Congenital portosystemic shunts (CPSSs) are often associated with life-threatening systemic complications, which may be detected by identifying hypergalactosemia in newborn screening (NBS). However, diagnosing CPSS at an early stage is not easy. The purpose of this study was to predict CPSS early using screening values and general blood tests. The medical records of 153 patients with hypergalactosemia who underwent NBS in Saitama Prefecture between 1 December 1997 and 31 October 2023 were retrospectively analyzed. We provided the final diagnosis of the analyzed patients. Of the 153 patients, 44 (29%) were in the CPSS group and 83 (54%) were in the transient galactosemia group. Using the initial screening items and the six blood test items, we attempted to extract a CPSS group from the transient galactosemia group. Finally, a model for CPSS prediction was established. From multiple logistic regression analysis, filtered blood galactose-1 phosphate/galactose, serum total bile acid, and ammonia were adopted as explanatory variables for the prediction model. If the cut-off value for predicted disease probability value (P) was >0.357, CPSS was identified with 86.4% sensitivity (95%CI 72.6–94.8%) and 81.9% specificity (95%CI 72.0–89.5%). This predictive model might allow prediction of CPSS and early intervention. Full article

(This article belongs to the Collection Newborn Screening in Japan)

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15 pages, 1371 KB

Open AccessSystematic Review

Refining CFTR-Related Metabolic Syndrome (CRMS)/Cystic Fibrosis Screen Positive, Inconclusive Diagnosis (CFSPID) Diagnosis: Impact of CFTR2 Variant Classifications

by MacKenzie Wyatt, Alexandra Quinn, Lincoln Shade and Meghan McGarry

Int. J. Neonatal Screen. 2025, 11(3), 60; https://doi.org/10.3390/ijns11030060 - 30 Jul 2025

Abstract

An unintended consequence of cystic fibrosis (CF) newborn screening (NBS) is the identification of infants with a positive NBS who do not meet the diagnostic criteria for CF (two CF-causing variants and/or sweat chloride > 60 mmol/L). This indeterminate diagnosis is called cystic [...] Read more.

An unintended consequence of cystic fibrosis (CF) newborn screening (NBS) is the identification of infants with a positive NBS who do not meet the diagnostic criteria for CF (two CF-causing variants and/or sweat chloride > 60 mmol/L). This indeterminate diagnosis is called cystic fibrosis transmembrane conductance regulator (CFTR)-related metabolic syndrome (CRMS) or CF screen positive, inconclusive diagnosis (CFSPID). CRMS/CFSPID occurs when it is not clearly known whether CFTR variants are disease-causing. In 2024, the CFTR2 classification of many CFTR variants was changed from unknown significance to either CF-causing variants or variants of varying clinical consequences (VVCCs). We conducted a meta-analysis of CRMS/CFSPID cases from manuscripts to describe how the diagnoses would change using two different variant panels: (1) only CF-causing CFTR variants (Panel_CF-causing) and (2) CF-causing variants and VVCCs (Panel_{CF-causing+VVCCs}). Using the Panel_CF-causing, 8.7% had two CF-causing variants (reclassified as CF), while 91.3% had less than two CF-causing variants (reclassified as Undetected). Using the Panel_{CF-causing+VVCCs}, 51.4% had either two VVCCs or one VVCC with one CF-causing variant detected (reclassified as CRMS/CFSPD), 39.9% had less than two CF-causing variants detected (reclassified as Undetected), and 8.7% had two CF-causing variants (reclassified as CF). In conclusion, using the updated CFTR2 classification of CFTR variants significantly decreases the number of children with CRMS/CFSPID and gives a definitive diagnosis of CF to some children while not detecting as many children who are unlikely to develop CF. Full article

(This article belongs to the Special Issue Advances in Cystic Fibrosis Newborn Screening: From Laboratory Testing to Diagnosis)

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14 pages, 882 KB

Open AccessArticle

Advancing Neonatal Screening for Pyridoxine-Dependent Epilepsy-ALDH7A1 Through Combined Analysis of 2-OPP, 6-Oxo-Pipecolate and Pipecolate in a Butylated FIA-MS/MS Workflow

by Mylène Donge, Sandrine Marie, Amandine Pochet, Lionel Marcelis, Geraldine Luis, François Boemer, Clément Prouteau, Samir Mesli, Matthias Cuykx, Thao Nguyen-Khoa, David Guénet, Aurélie Empain, Magalie Barth, Benjamin Dauriat, Cécile Laroche-Raynaud, Corinne De Laet, Patrick Verloo, An I. Jonckheere, Manuel Schiff, Marie-Cécile Nassogne and Joseph P. Dewulf Show full author list Hide full author list

Int. J. Neonatal Screen. 2025, 11(3), 59; https://doi.org/10.3390/ijns11030059 - 30 Jul 2025

Abstract

Pyridoxine-dependent epilepsy (PDE) represents a group of rare developmental and epileptic encephalopathies. The most common PDE is caused by biallelic pathogenic variants in ALDH7A1 (PDE-ALDH7A1; OMIM #266100), which encodes α-aminoadipate semialdehyde (α-AASA) dehydrogenase, a key enzyme in lysine catabolism. Affected individuals present with [...] Read more.

Pyridoxine-dependent epilepsy (PDE) represents a group of rare developmental and epileptic encephalopathies. The most common PDE is caused by biallelic pathogenic variants in ALDH7A1 (PDE-ALDH7A1; OMIM #266100), which encodes α-aminoadipate semialdehyde (α-AASA) dehydrogenase, a key enzyme in lysine catabolism. Affected individuals present with seizures unresponsive to conventional anticonvulsant medications but responsive to high-dose of pyridoxine (vitamin B6). Adjunctive lysine restriction and arginine supplementation have also shown potential in improving neurodevelopmental outcomes. Given the significant benefit of early intervention, PDE-ALDH7A1 is a strong candidate for newborn screening (NBS). However, traditional biomarkers are biochemically unstable at room temperature (α-AASA and piperideine-6-carboxylate) or lack sufficient specificity (pipecolate), limiting their utility for biomarker-based NBS. The recent identification of two novel and stable biomarkers, 2S,6S-/2S,6R-oxopropylpiperidine-2-carboxylate (2-OPP) and 6-oxo-pipecolate (oxo-PIP), offers renewed potential for biochemical NBS. We evaluated the feasibility of incorporating 2-OPP, oxo-PIP, and pipecolate into routine butylated FIA-MS/MS workflows used for biochemical NBS. A total of 9402 dried blood spots (DBS), including nine confirmed PDE-ALDH7A1 patients and 9393 anonymized controls were analyzed using a single multiplex assay. 2-OPP emerged as the most sensitive biomarker, identifying all PDE-ALDH7A1 patients with 100% sensitivity and a positive predictive value (PPV) of 18.4% using a threshold above the 99.5th percentile. Combining elevated 2-OPP (above the 99.5th percentile) with either pipecolate or oxo-PIP (above the 85.0th percentile) as secondary marker detected within the same multiplex FIA-MS/MS assay further improved the PPVs to 60% and 45%, respectively, while maintaining compatibility with butanol-derivatized method. Notably, increasing the 2-OPP threshold above the 99.89th percentile, in combination with either pipecolate or oxo-PIP above the 85.0th percentile resulted in both 100% sensitivity and 100% PPV. This study supports the strong potential of 2-OPP-based neonatal screening for PDE-ALDH7A1 within existing NBS infrastructures. The ability to multiplex 2-OPP, pipecolate and oxo-PIP within a single assay offers a robust, practical, high-throughput and cost-effective approach. These results support the inclusion of PDE-ALDH7A1 in existing biochemical NBS panels. Further prospective studies in larger cohorts are needed to refine cutoffs and confirm clinical performance. Full article

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10 pages, 1202 KB

Open AccessArticle

Incidence of Congenital Hypothyroidism Is Increasing in Chile

by Francisca Grob, Gabriel Cavada, Gabriel Lobo, Susana Valdebenito, Maria Virginia Perez and Gilda Donoso

Int. J. Neonatal Screen. 2025, 11(3), 58; https://doi.org/10.3390/ijns11030058 - 26 Jul 2025

Abstract

Congenital hypothyroidism (CH) is a leading preventable cause of neurocognitive impairment. Its incidence appears to be rising in several countries. We analysed 27 years of newborn-screening data (1997–2023) from the largest Chilean screening centre, covering 3,225,216 newborns (51.1% of national births), to characterise [...] Read more.

Congenital hypothyroidism (CH) is a leading preventable cause of neurocognitive impairment. Its incidence appears to be rising in several countries. We analysed 27 years of newborn-screening data (1997–2023) from the largest Chilean screening centre, covering 3,225,216 newborns (51.1% of national births), to characterise temporal trends and potential drivers of CH incidence. Annual CH incidence was modelled with Prais–Winsten regression to correct for first-order autocorrelation; additional models assessed trends in gestational age, sex, biochemical markers, and aetiological subtypes. We identified 1550 CH cases, giving a mean incidence of 4.9 per 10,000 live births and a significant yearly increase of 0.067 per 10,000 (95 % CI 0.037–0.098; p < 0.001). Mild cases (confirmation TSH < 20 mU/L) rose (+0.89 percentage points per year; p = 0.002). The program’s recall was low (0.05%). Over time, screening and diagnostic TSH values declined, total and free T4 concentrations rose, gestational age at diagnosis fell, and a shift from thyroid ectopy toward hypoplasia emerged; no regional differences were detected. The sustained increase in CH incidence, alongside falling TSH thresholds and growing detection of in situ glands, suggests enhanced recognition of milder disease. Ongoing surveillance should integrate environmental, iodine-nutrition, and genetic factors to clarify the causes of this trend. Full article

(This article belongs to the Special Issue Newborn Screening for Congenital Hypothyroidism)

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International Journal of Neonatal Screening

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