Recommended Uniform Screening Panel Research Articles

Krabbe Disease–To Add or Not to Newborn Screening?

Krabbe disease (KD), a severe neurodegenerative disorder, has been controversial in the space of newborn screening (NBS) in the United States. Families continue to advocate for the addition of KD to the Recommended Uniform Screening Panel (RUSP) after being declined for the second time in February 2023. Even with significant progress in KD screening tests, uncertainty about the phenotypic presentations, and effectiveness of hematopoietic stem cell transplant (HSCT) seems to have impeded the addition of this condition to the RUSP. Potential in-utero onset of symptoms in early infantile onset Krabbe disease (EIKD) raise questions on the ‘pre-symptomatic’ requirement of NBS. This paper reviews the current knowledge of KD, including accepted and investigational treatments to help further the discussion for adding KD on NBS panels.

NBSTRN Tools to Advance Newborn Screening Research and Support Newborn Screening Stakeholders.

Rapid advances in the screening, diagnosis, and treatment of genetic disorders have increased the number of conditions that can be detected through universal newborn screening (NBS). However, the addition of conditions to the Recommended Uniform Screening Panel (RUSP) and the implementation of nationwide screening has been a slow process taking several years to accomplish for individual conditions. Here, we describe web-based tools and resources developed and implemented by the newborn screening translational research network (NBSTRN) to advance newborn screening research and support NBS stakeholders worldwide. The NBSTRN's tools include the Longitudinal Pediatric Data Resource (LPDR), the NBS Condition Resource (NBS-CR), the NBS Virtual Repository (NBS-VR), and the Ethical, Legal, and Social Issues (ELSI) Advantage. Research programs, including the Inborn Errors of Metabolism Information System (IBEM-IS), BabySeq, EarlyCheck, and Family Narratives Use Cases, have utilized NBSTRN's tools and, in turn, contributed research data to further expand and refine these resources. Additionally, we discuss ongoing tool development to facilitate the expansion of genetic disease screening in increasingly diverse populations. In conclusion, NBSTRN's tools and resources provide a trusted platform to enable NBS stakeholders to advance NBS research and improve clinical care for patients and their families.

Variant Classification for Pompe disease; ACMG/AMP specifications from the ClinGen Lysosomal Diseases Variant Curation Expert Panel

Accurate determination of the clinical significance of genetic variants is critical to the integration of genomics in medicine. To facilitate this process, the NIH-funded Clinical Genome Resource (ClinGen) has assembled Variant Curation Expert Panels (VCEPs), groups of experts and biocurators which provide gene- and disease- specifications to the American College of Medical Genetics & Genomics and Association for Molecular Pathology's (ACMG/AMP) variation classification guidelines. With the goal of classifying the clinical significance of GAA variants in Pompe disease (Glycogen storage disease, type II), the ClinGen Lysosomal Diseases (LD) VCEP has specified the ACMG/AMP criteria for GAA. Variant classification can play an important role in confirming the diagnosis of Pompe disease as well as in the identification of carriers. Furthermore, since the inclusion of Pompe disease on the Recommended Uniform Screening Panel (RUSP) for newborns in the USA in 2015, the addition of molecular genetic testing has become an important component in the interpretation of newborn screening results, particularly for asymptomatic individuals. To date, the LD VCEP has submitted classifications and supporting data on 243 GAA variants to public databases, specifically ClinVar and the ClinGen Evidence Repository. Here, we describe the ACMG/AMP criteria specification process for GAA, an update of the GAA-specific variant classification guidelines, and comparison of the ClinGen LD VCEP's GAA variant classifications with variant classifications submitted to ClinVar. The LD VCEP has added to the publicly available knowledge on the pathogenicity of variants in GAA by increasing the number of expert-curated GAA variants present in ClinVar, and aids in resolving conflicting classifications and variants of uncertain clinical significance.

Multiplexing Iduronate-2-Sulphatase (MPS-II) into a 7-Plex Lysosomal Storage Disorder MS/MS Assay Using Cold-Induced Phase Separation.

Mucopolysaccharidosis type II (MPS-II, Hunter syndrome, OMIM:30990) is a lysosomal storage disorder (LSD) that results in iduronate 2-sulphatase (I2S) enzyme deficiency. MPS-II was added to the Recommended Uniform Screening Panel (RUSP) in August 2022; thus, there is an increased demand for multiplexing I2S into existing LSD screening assays. After incubation with LSD synthetic substrates, extracts are cleaned using liquid-liquid extraction with ethyl acetate or protein precipitation using acetonitrile (ACN). We investigated cold-induced water ACN phase separation (CIPS) to improve the combination of 6-plex and I2S extracts to create a 7-plex assay, and compared it to room temperature ACN and ethyl acetate liquid-liquid extraction. The extracts were dried and resuspended in the mobile phase, and then analyzed using an optimized 1.9 min injection-to-injection liquid chromatography method coupled with tandem mass spectrometry (LC-MS/MS). The combination of ACN and CIPS improved the detection for I2S products without significant detriment to other analytes, which is attributable to a more complete coagulation and separation of heme, proteins, and extracted residual salts. Using CIPS for sample cleanup in dried blood spots (DBS) appears to represent a promising and straightforward way of achieving cleaner sample extracts in a new 7-plex LSD screening panel.

Infants with Congenital Diseases Identified through Newborn Screening—United States, 2018–2020

Newborn screening (NBS) is a state or territory-based public health system that screens newborns for congenital diseases that typically do not present with clinical symptoms at birth but can cause significant mortality and morbidity if not detected or treated quickly. NBS continues to be one of the most successful public health interventions in the US, providing early detection and intervention to all infants. The increase in overall birth prevalence of core Recommended Uniform Screening Panel (RUSP) diseases detected via dried blood spot (DBS) specimens from 2015-2017 (17.50-18.31 per 10,000) to 2018-2020 (20.07 per 10,000), as reported into the APHL NewSTEPs database, affirms the importance and impact of NBS programs. This report presents aggregate numbers and birth prevalence of diseases detected by DBS on the RUSP from 2018-2020, including data from fifty US states and two territories.

Implementation of Newborn Screening for Conditions in the United States First Recommended during 2010-2018.

The Recommended Uniform Screening Panel (RUSP) is the list of conditions recommended by the US Secretary of Health and Human Services for inclusion in state newborn screening (NBS). During 2010-2022, seven conditions were added to the RUSP: severe combined immunodeficiency (SCID) (2010), critical congenital heart disease (CCHD) (2011), glycogen storage disease, type II (Pompe) (2015), mucopolysaccharidosis, type I (MPS I) (2016), X-linked adrenoleukodystrophy (X-ALD) (2016), spinal muscular atrophy (SMA) (2018), and mucopolysaccharidosis, type II (MPS II) (2022). The adoption of SCID and CCHD newborn screening by programs in all 50 states and three territories (Washington, D.C.; Guam; and Puerto Rico) took 8.6 and 6.8 years, respectively. As of December 2022, 37 programs screen for Pompe, 34 for MPS I, 32 for X-ALD, and 48 for SMA. The pace of implementation based on the average additional number of NBS programs per year was most rapid for SMA (11.3), followed by CCHD (7.8), SCID (6.2), MPS I (5.4), Pompe (4.9), and X-ALD (4.7).

Validation of a targeted metabolomics panel for improved second-tier newborn screening.

Improved second-tier assays are needed to reduce the number of false positives in newborn screening (NBS) for inherited metabolic disorders including those on the Recommended Uniform Screening Panel (RUSP). We developed an expanded metabolite panel for second-tier testing of dried blood spot (DBS) samples from screen-positive cases reported by the California NBS program, consisting of true- and false-positives from four disorders: glutaric acidemia type I (GA1), methylmalonic acidemia (MMA), ornithine transcarbamylase deficiency (OTCD), and very long-chain acyl-CoA dehydrogenase deficiency (VLCADD). This panel was assembled from known disease markers and new features discovered by untargeted metabolomics and applied to second-tier analysis of single DBS punches using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a 3-min run. Additionally, we trained a Random Forest (RF) machine learning classifier to improve separation of true- and false positive cases. Targeted metabolomic analysis of 121 analytes from DBS extracts in combination with RF classification at a sensitivity of 100% reduced false positives for GA1 by 83%, MMA by 84%, OTCD by 100%, and VLCADD by 51%. This performance was driven by a combination of known disease markers (3-hydroxyglutaric acid, methylmalonic acid, citrulline, and C14:1), other amino acids and acylcarnitines, and novel metabolites identified to be isobaric to several long-chain acylcarnitine and hydroxy-acylcarnitine species. These findings establish the effectiveness of this second-tier test to improve screening for these four conditions and demonstrate the utility of supervised machine learning in reducing false-positives for conditions lacking clearly discriminating markers, with future studies aimed at optimizing and expanding the panel to additional disease targets.

A Roadmap for Potential Improvement of Newborn Screening for Inherited Metabolic Diseases Following Recent Developments and Successful Applications of Bivariate Normal Limits for Pre-Symptomatic Detection of MPS I, Pompe Disease, and Krabbe Disease.

The mucopolysaccharidoses (MPS), Pompe Disease (PD), and Krabbe disease (KD) are inherited conditions known as lysosomal storage disorders (LSDs) The resulting enzyme deficiencies give rise to progressive symptoms. The United States Department of Health and Human Services' Recommended Uniform Screening Panel (RUSP) suggests LSDs for inclusion in state universal newborn screening (NBS) programs and has identified screening deficiencies in MPS I, KD, and PD NBS programs. MPS I NBS programs utilize newborn dried blood spots and assay alpha L-iduronidase (IDUA) enzyme to screen for potential cases. Glycosaminoglycans (GAGs) offer potential as a confirmatory test. KD NBS programs utilize galactocerebrosidase (GaLC) as an initial test, with psychosine (PSY) activity increasingly used as a confirmatory test for predicting onset of Krabbe disease, though with an excessive false positive rate. PD is marked by a deficiency in acid α-glucosidase (GAA), causing increased glycogen, creatine (CRE), and other biomarkers. Bivariate normal limit (BVNL) methods have been applied to GaLC and PSY activity to produce a NBS tool for KD, and more recently, to IDUA and GAG activity to develop a NBS tool for MPS I. A BVNL tool based on GAA and CRE is in development for infantile PD diagnosis. Early infantile KD, MPS I, and PD cases were pre-symptomatically identified by BVNL-based NBS tools. This article reviews these developments, discusses how they address screening deficiencies identified by the RUSP and may improve NBS more generally.

DbRUSP: An Interactive Database to Investigate Inborn Metabolic Differences for Improved Genetic Disease Screening.

The Recommended Uniform Screening Panel (RUSP) contains more than forty metabolic disorders recommended for inclusion in universal newborn screening (NBS). Tandem-mass-spectrometry-based screening of metabolic analytes in dried blood spot samples identifies most affected newborns, along with a number of false positive results. Due to their influence on blood metabolite levels, continuous and categorical covariates such as gestational age, birth weight, age at blood collection, sex, parent-reported ethnicity, and parenteral nutrition status have been shown to reduce the accuracy of screening. Here, we developed a database and web-based tools (dbRUSP) for the analysis of 41 NBS metabolites and six variables for a cohort of 500,539 screen-negative newborns reported by the California NBS program. The interactive database, built using the R shiny package, contains separate modules to study the influence of single variables and joint effects of multiple variables on metabolite levels. Users can input an individual’s variables to obtain metabolite level reference ranges and utilize dbRUSP to select new candidate markers for the detection of metabolic conditions. The open-source format facilitates the development of data mining algorithms that incorporate the influence of covariates on metabolism to increase accuracy in genetic disease screening.

Newborn screening for Pompe disease: Parental experiences and follow-up care for a late-onset diagnosis.

Newborn screening (NBS) for Pompe disease (PD) was added to the Recommended Uniform Screening Panel (RUSP) in the United States in 2015 because there was compelling evidence of health benefits for early diagnosis of Infantile-onset Pompe disease (IOPD). However, one limitation of NBS for PD is its inability to distinguish IOPD and late onset forms of Pompe disease (LOPD). Management of LOPD is challenging because of uncertainty around progression of LOPD and determining the appropriate time for treatment initiation. The aims of this study were to understand the impact of LOPD identified through NBS, by exploring the differences in attitudes, emotions and opinions among parents and identify their needs for follow-up care. Study participants were recruited from states that included PD on their NBS panel. Semi-structured interviews were conducted with parents of nine children who were diagnosed with LOPD after an abnormal NBS result. Predominantly, parents reported a lack of adequate information, guidance, and psychosocial support from the very beginning and through the course of their diagnosis. This caused uncertainty, anxiety, frustration, and fear of the unknown. Parents live in a 'worry or not to worry' phase, balancing between coping methods to avoid over medicalization of their child, but also preparing concrete follow-up plans to be on the lookout for any signs of PD-related symptoms. Understanding parents' experiences allows genetic counselors and NBS programs to proactively design care plan for parents during this difficult period.

Newborn Screening for Spinal Muscular Atrophy in New York State: Clinical Outcomes From the First 3 Years.

Spinal muscular atrophy (SMA) was added to the Recommended Uniform Screening Panel in July 2018 largely on the basis of the availability and efficacy of newly approved disease-modifying therapies. New York State (NYS) started universal newborn screening for SMA in October 2018. The authors report the findings from the first 3 years of screening. Statewide neonatal screening was conducted using DNA extracted from dried blood spots using a real-time quantitative PCR assay. Retrospective follow-up data were collected from 9 referral centers across the state on 34 infants. In the first 3 years since statewide implementation, nearly 650,000 infants have been screened for SMA. Thirty-four babies screened positive and were referred to a neuromuscular specialty care center. The incidence remains lower than previously predicted. The majority (94%), including all infants with 2-3 copies of survival motor neuron (SMN) 2, have received treatment. Among treated infants, the overwhelming majority (94%; 30/32) have received gene replacement. All infants in this cohort with 3 copies of SMN2 are clinically asymptomatic posttreatment based on early clinical follow-up data. Infants with 2 copies of SMN2 are more variable in their outcomes. Electrodiagnostic outcomes data obtained from a subgroup of patients (n = 11) demonstrated either improvement or no change in compound muscle action potential (CMAP) amplitude at last clinical follow-up compared with pretreatment baseline. Most infants were treated before 6 weeks of age (median = 34.5 days of life; range 11-180 days). Delays and barriers to treatment identified by treating clinicians followed 2 broad themes: medical and nonmedical. Medical delays most commonly reported were the presence of AAV9 antibodies and elevated troponin I levels. Nonmedical barriers included delays in obtaining insurance and insurance policies regarding specific treatment modalities. The findings from the NYS cohort of newborn screen-identified infants are consistent with other reports of improved outcomes from early diagnosis and treatment. Additional biomarkers of motor neuron health including EMG can potentially be helpful in detecting preclinical decline.

Newborn screening and the recommended uniform screening panel: Optimal submissions and suggested improvements based on an advocacy organization's decade-long experience.

The National MPS Society, Inc., founded in 1974, is a rare disease advocacy non-profit with a tripartite mission addressing the needs of the mucopolysaccharidosis and mucolipidosis communities through advocacy, research, and family and patient support. The Recommended Uniform Screening Panel (RUSP) of conditions for newborn screening (NBS), legislatively mandated in 2008, was implemented in 2010 by the Secretary of Health and Human Services (HSS), through the adoption of 29 core conditions. Since its inception the RUSP has grown to 35 core conditions. Each addition followed a defined nomination process that has itself undergone further definition over time. Since the adoption of the RUSP, the Society has nominated two conditions that have been approved by the Advisory Committee on Heritable Disorders in Children and Newborns (ACHDNC) and forwarded to the Secretary of HSS for inclusion on the RUSP. This history places the Society in a position to reflect on the process of successfully nominating conditions. Additionally, the Society is well placed by this experience to provide observations on the RUSP process. We will highlight best practices for pending and future nominations and reflect on potential improvements to the process and infrastructure of NBS, the RUSP, and the ACHDNC.

Why must the debate continue on Krabbe disease newborn screening?

Since the early 2000s, many families impacted by Krabbe disease have tried to implement newborn screening for this rare fatal neurological disorder in their home state. However, despite grassroots efforts, states have been unable to agree to newborn screening for Krabbe disease due to poor testing mechanisms, lack of understanding of the developmental outcomes of transplantation, low incidence rate, and more. Over the past five years, many organizations and experts have made significant strides to help Krabbe disease meet the eligibility requirements for state panels and the Recommended Uniform Screening Panel (RUSP). Nevertheless, ethicists and newborn screening advisory committees continue to disregard the progress our community has made in the treatment and screening of Krabbe disease.

EP008: Cystathionine beta synthase deficiency patients are being missed by newborn screening

Newborn screening for Cystathionine beta Synthase (CBS deficiency, also known as Homocystinuria) was initiated in some states as early as 1968 with the aim of preventing devastating complications from inadequate early treatment. During the 1990s, improvements in technology allowed for CBS deficiency to be detected by mass spectrometry detection of methionine levels from the dried blood spot card. Not until 2009 was CBS deficiency recommended to be universally screened in all US states following its inclusion in the Recommended Uniform Screening Panel (RUSP).

EP504: A novel visualization of state-by-state variability in newborn screening highlights underrepresentation of lysosomal storage disorders

Newborn screening (NBS) is an important, cost-effective tool that can detect dozens of early-onset and treatable disorders in the approximately 3.6 million newborns who are screened in the US each year. The disorders covered on the NBS vary from state to state, and no two states screen for the same set of disorders. The state-level factors associated with the disorders covered on a particular state’s NBS have not been systematically investigated, even though this knowledge could have important implications for state and national NBS policy. Furthermore, the display of state-to-state NBS data in a visual format could provide a quick reference for policymakers to identify gaps in categories of disorders that are systematically underrepresented on the NBS at a national level. Publicly-available data on the disorders listed on the NBS in each state was obtained from www.babysfirsttest.org and www.newsteps.org, websites that curate state-level data on newborn screening, in Spring 2021. Disorders were then grouped into one of seven categories (eg, endocrine, amino acid, organic acid, fatty acid oxidation, lysosomal storage, hemoglobin, and other). Data was also sourced from publicly-available sources such as https://www.census.gov/ that contain state-level variables on population size and demographics. Data were collected using the program rvest (version 0.3.6) and were analyzed and visualized using ggplot2 (version 3.3.5) in the statistical programming language R (version 4.0.3). The resulting heatmap visualization of NBS contains disorders on the x-axis and states on the y-axis, with the value within each cell indicating whether a given state screens for a particular disorder. The NBS in Tennessee (67 disorders at the time of analysis), the state with the most conditions screened for, contains more than double the number of conditions screened for compared to Arizona, the state with the fewest conditions screened for (31 conditions). In general, states that have larger populations also tend to screen for more disorders on their NBS. In addition, 30 of the 36 primary disorders listed on the July 2018 version of the Recommended Uniform Screening Panel (RUSP), a national guideline of the minimum recommended conditions that should be present on a state’s NBS, were listed on the NBS for all states. Furthermore, the data highlight that few states are screening for lysosomal storage disorders (LSDs). In particular, Michigan, Tennessee, and Missouri are the only states to include 5 or more LSDs on their NBS panel, with over half of all states not including any LSDs on their NBS. This novel heatmap visualization highlights the striking variability present in NBS offerings between states. Summary statistics and an overview of the state-to-state variation in newborn screening in the U.S. can be rapidly ascertained from this visualization in a manner that was not previously possible. In particular, this visualization clearly demonstrates that lysosomal storage diseases are systematically underrepresented in newborn screening nationwide, despite the increasing availability of enzyme replacement therapies for this class of disorders. This publicly-available visualization of NBS will more easily allow advocates for particular disorders to identify gaps in NBS nationwide and within particular states.

Newborn Screening Practices for Beta-Thalassemia in the United States.

Beta-thalassemia, a heritable condition of abnormal hemoglobin production, is not a core condition on the United States Recommended Uniform Screening Panel (RUSP) for state and territorial newborn screening (NBS) programs. However, screening for sickle cell disease (which is on the core RUSP) also detects reduced or absent levels of hemoglobin (Hb) A and certain other Hb variants associated with beta-thalassemia and, thus, allows for a timely referral to appropriate healthcare to minimize sequalae of the disease. The Association of Public Health Laboratories’ Hemoglobinopathy Workgroup administered a comprehensive survey of all U.S. NBS programs to assess beta-thalassemia testing methodologies, the cutoffs for defining beta-thalassemia major, and the reporting and follow-up practices. Forty-six (87%) of the programs responded. Thirty-nine of the 46 responding programs (85%) report some form of suspected beta-thalassemia; however, the screening methods, the percentage of Hb A used as a cutoff for an indication of beta-thalassemia major, and the screening follow-up vary widely. The standardization of technical and reporting procedures may improve access to specialty care prior to severe complications, increase genetic counseling, and provide data needed to better understand the public health impact and clinical outcomes of beta-thalassemia in the United States.

Prospective diagnosis of MT-ATP6-related mitochondrial disease by newborn screening

Elevated citrulline and C5-OH levels are reported as part of the newborn screening of core and secondary disorders on the Recommended Uniform Screening Panel (RUSP). Additionally, some state laboratory newborn screening programs report low citrulline levels, which may be observed in proximal urea cycle disorders. We report six patients who were found on newborn screening to have low citrulline and/or elevated C5-OH levels in whom confirmatory testing showed the combination of these two abnormal analytes. Mitochondrial sequencing revealed known pathogenic variants in MT-ATP6 at high heteroplasmy levels in all cases. MT-ATP6 at these heteroplasmy levels is associated with Leigh syndrome, a progressive neurodegenerative disease. Patients were treated with supplemental citrulline and, in some cases, mitochondrial cofactor therapy. These six patients have not experienced metabolic crises or developmental regression, and early diagnosis and management may help prevent the neurological sequelae of Leigh syndrome. The affected mothers and siblings are asymptomatic or paucisymptomatic (e.g. intellectual disability, depression, migraines, obsessive-compulsive disorder, and poor balance) despite high heteroplasmy or apparent homoplasmy of the familial variant, thus expanding the clinical spectrum seen in pathogenic variants of MT-ATP6. Confirmatory plasma amino acid analysis and acylcarnitine profiling should be ordered in a patient with either low citrulline and/or elevated C5-OH, as this combination appears specific for pathogenic variants in MT-ATP6.

Screen for Lysosomal Storage Diseases, Save More Nevada Babies

IntroductionIn the US, the Recommended Uniform Screening Panel (RUSP) currently endorses 29 disorders for which newborns should be screened, given evidence of the benefits of early detection. With ...

Mucopolysaccharidosis type I newborn screening: Importance of second tier testing for ethnically diverse populations.

Mucopolysaccharidosis type I (MPS I)/Hurler syndrome newborn screening was added to the recommended uniform screening panel (RUSP) in 2016. As states have added screening for MPS I, programs have reported increased rates of false positives. Reasons for false positive screens include carrier status, true false positive, late-onset/attenuated forms, and in about half of cases, pseudodeficiency alleles. These alleles have DNA variants that can cause falsely decreased enzyme activity on biochemical enzyme studies and have increased frequency in individuals of African American and African descent. We describe the District of Columbia (DC) experience with MPS I screening from December 2017 to February 2019. In the context of a review of the literature on newborn screening and family experiences and this DC-based experience, we offer potential solutions to address preliminary concerns regarding this screening. The impact of overrepresentation of screen positives in a minority group and unintentional creation of health disparities and community wariness regarding medical genetics evaluations must be considered to improve the newborn screen programs nationally and internationally.

Translating Molecular Technologies into Routine Newborn Screening Practice.

As biotechnologies advance and better treatment regimens emerge, there is a trend toward applying more advanced technologies and adding more conditions to the newborn screening (NBS) panel. In the current Recommended Uniform Screening Panel (RUSP), all conditions but one, congenital hypothyroidism, have well-defined genes and inheritance patterns, so it is beneficial to incorporate molecular testing in NBS when it is necessary and appropriate. Indeed, the applications of molecular technologies have taken NBS to previously uncharted territory. In this paper, based on our own program experience and what has been reported in the literature, we describe current practices regarding the applications of molecular technologies in routine NBS practice in the era of genomic and precision medicine.