Newborn screening for inborn errors of metabolism

INTRODUCTION — Newborn screening (NBS) programs exist in many countries worldwide. The goal of NBS is to detect readily treatable disorders that are threatening to life or long-term health before they become symptomatic. Early initiation of treatment may prevent or significantly reduce mortality and morbidity in affected patients, making screening programs using a high-throughput, low-cost screening test with high sensitivity and specificity an important and cost-effective public health measure. Many inborn errors of metabolism (IEM) meet criteria for inclusion in NBS programs.

The rationale and tests available for NBS for IEM are reviewed here. The general principles of NBS, screening policies, testing, and follow-up are discussed in detail separately. (See "Overview of newborn screening".)

RECOMMENDED UNIFORM SCREENING PANEL (RUSP)

Overview of RUSP — NBS is possible for more than 50 IEM, but each state in the United States determines which conditions are included in the program within their jurisdiction. The US Health Resources and Services Administration established the Recommended Uniform Screening Panel (RUSP), a list of conditions that all NBS programs should include, in order to minimize variability across states. However, each state has their own process for the nomination and adoption of new conditions to their programs. Several states require RUSP nomination by law to add a new condition. (See "Overview of newborn screening", section on 'Programs throughout the world' and "Overview of newborn screening", section on 'Implementation of screening in the United States'.)

The RUSP list includes both core conditions that every newborn should be screened for and secondary conditions that can be detected when screening for a core condition but that do not qualify as such due to lack of effective treatment. As an example, phenylketonuria (PKU) is a core condition. Screening for it by measurement of phenylalanine also leads to the identification of patients with disorders of tetrahydrobiopterin synthesis and recycling. Tetrahydrobiopterin is a cofactor of phenylalanine, tyrosine, and tryptophan hydroxylases, and its absence therefore causes hyperphenylalaninemia but also deficiency of the neurotransmitters dopamine and serotonin, resulting in severe and difficult-to-treat neurologic diseases [1].

Metabolic disorders included on the RUSP — Most disorders on the RUSP are IEM and include disorders of amino acid, carbohydrate, fatty acid, and organic acid metabolism and transport. In 2010, when the RUSP became a federal recommendation, it listed 22 metabolic diseases as core conditions and 24 metabolic secondary target conditions. Of these, 20 and 22, respectively, are identified by simultaneous analysis of acylcarnitines and amino acids from a single 3 mm dried blood spot (DBS) punch. Lysosomal acid alpha-glucosidase deficiency (Pompe disease, glycogen storage disease II, acid maltase deficiency) was added in March 2015. Mucopolysaccharidosis type I (MPS I) and the peroxisomal disorder X-linked adrenoleukodystrophy (X-ALD) were added in February 2016 [2]. A third lysosomal disorder, mucopolysaccharidosis type II (MPS II), was added in August 2022, bringing the number of metabolic core conditions included in the RUSP to 26. (See 'NBS for specific IEM' below.)

Nonmetabolic disorders included on the RUSP — The nonmetabolic conditions included in the RUSP are congenital hypothyroidism, congenital adrenal hyperplasia, sickle cell disease and other hemoglobinopathies, cystic fibrosis, severe combined immunodeficiencies, spinal muscular atrophy, hearing loss, and critical congenital cyanotic heart disease. The last two conditions are not screened by testing of DBS but at the bedside with brainstem auditory evoked response (BAER) and pulse oximetry, respectively. (See "Screening the newborn for hearing loss" and "Newborn screening for inborn errors of immunity" and "Newborn screening for critical congenital heart disease using pulse oximetry" and "Diagnosis of sickle cell disorders", section on 'Newborn screening' and "Clinical features and detection of congenital hypothyroidism", section on 'Newborn screening' and "Cystic fibrosis: Clinical manifestations and diagnosis", section on 'Newborn screening' and "Clinical manifestations and diagnosis of classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency in infants and children", section on 'Newborn screening' and "Spinal muscular atrophy", section on 'Diagnosis'.)

OVERVIEW OF NBS

Conditions included and testing approaches — The conditions included on NBS panels and the actual laboratory tests and testing strategies employed by NBS laboratories vary from program to program. Except for the bedside tests for hearing loss and critical congenital cyanotic heart disease, all NBS relies on newborn blood samples collected on filter paper (dried blood spots [DBS] on Guthrie cards). Factors that programs need to consider before adding a new condition to their panels include the amount of blood available on the Guthrie card and whether the analysis of the relevant screening marker(s) can be combined with existing tests or methods. Screening strategies should also include ensuring low false-positive rates while accurately identifying affected patients that need attention in a timely fashion. (See "Overview of newborn screening".)

Phenylketonuria (PKU), an inborn error of amino acid metabolism, was the condition for which NBS was first developed in the early 1960s by Dr. Robert Guthrie [3]. Guthrie's original laboratory approach relied on a bacterial inhibition assay specific to phenylalanine. NBS programs initially required a dedicated test and DBS punch for each new condition added to the program. Tandem mass spectrometry (MS/MS), introduced in the late 1990s, quickly led to an expansion of NBS menus because MS/MS allows for the simultaneous and rapid analysis of multiple markers (such as amino acids, acylcarnitines, and succinylacetone) in a single DBS punch. MS/MS not only allowed for more efficient laboratory testing but also enabled improvements in sensitivity and specificity [4-13]. The development and availability of an increasing number of therapies for more IEM that benefit from early intervention have also led to new DBS-based screening tests, often using the MS/MS platform.

Initial screening tests — Blood from the newborn is collected on filter paper (usually on the second day of life) and is sent for analysis. For most tests, if an abnormal result is detected, the next step in the process is communication of the test results to the primary care clinician of record, who then notifies the parent(s)/caregiver(s) of the newborn.

Second-tier tests performed on the original NBS sample can be employed to clarify abnormal results for primary screening markers with poor specificity (overlapping reference and disease ranges) and determine if a screening result is reported as positive or negative. However, these tests are not available for most primary analytes, and only a few programs make use of these tests.

Timeliness guidelines have been established in the United States, specifically that positive results for critical conditions should be reported by the NBS program to the clinician by the fifth day of life [14]. Many NBS programs have adjusted their business and holiday hours to meet this recommendation [15].

Role of the primary care clinician — Primary care clinicians should familiarize themselves with the NBS program(s) in their region and the relevant disease experts for consultations and be prepared to act quickly when receiving an abnormal screening result on one of their newborn patients. Depending upon the disorder suggested by the NBS result, the family may need to be contacted immediately to check on the infant's clinical state and then determine whether the infant can be seen in clinic or must be transported to an emergency department. Most conditions, however, can initially be managed in the clinic where confirmatory testing and preliminary treatment are initiated in consultation with a specialist.

Primary care clinicians also must remain vigilant and aware that some IEM, such as medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency, can manifest in the first few days of life prior to availability of NBS results, with potentially fatal outcomes if not recognized and addressed (table 1) [16].

When to refer — All newborns with abnormal NBS results should be referred to a specialist or specialty center when a diagnosis has been established at the latest. However, immediate consultation and referral are indicated for those neonates with an abnormal screen that are already symptomatic or are clinically asymptomatic but have a high likelihood of being affected with a critical condition (table 1). In some states, NBS programs inform the specialist proactively and in parallel to reporting a presumptive positive result to the primary care clinician.

ACT Sheets and algorithms — The American College of Medical Genetics and Genomics (ACMG) has available on their website one-page action sheets (ACT Sheets) and laboratory testing algorithms to inform clinicians about the implications of a screening result, the urgency of follow-up, as well as the basic and specialized laboratory tests that are necessary to clarify a potential differential diagnosis or identify a false-positive screening result (table 1).

Sensitivity and specificity — The sensitivity and specificity of each test is not well known, because screening programs rarely share this information and, if they do, it is mostly in aggregate for the whole program rather than by test or analyte. It also is dependent on the interpretation of results by the screening laboratories and where the cutoff for each analyte is set.

Short- and long-term follow-up — Short-term follow-up is concluded when a final diagnosis has been established after a presumptive positive screening result was reported. Most programs make a dedicated effort to ensure that patients are followed up at least to this point and in a timely fashion. In the US, the Association of Public Health Laboratories (APHL) Newborn Screening Technical Assistance and Evaluation Program (NewSTEPs) captures short-term follow-up data among its quality indicators.

It has been a longstanding assumption or understanding that long-term follow-up is the responsibility of clinicians rather than the NBS program [17]. However, the Advisory Committee on Heritable Disorders in Newborns and Children (ACHDNC) has worked to address long-term follow-up in order to ensure that patients identified through NBS receive appropriate care throughout their lifetime [18,19].

NBS FOR SPECIFIC IEM

Aminoacidopathies — NBS for aminoacidopathies began with phenylketonuria (PKU) in the 1960s. Since then, additional screening tests have been added, including for maple syrup urine disease (MSUD) and several urea cycle disorders that benefit from early identification because life-threatening neonatal ketoacidosis or hyperammonemia, respectively, can be prevented or more successfully treated when diagnosed early (table 1). (See "Overview of phenylketonuria", section on 'Diagnosis' and "Overview of phenylketonuria", section on 'Diagnosis of BH4 deficiency' and "Urea cycle disorders: Clinical features and diagnosis", section on 'Newborn screening'.)

Fatty acid oxidation disorders — Mitochondrial fatty acid oxidation is an essential pathway for energy production. It is required during prolonged fasting and/or increased energy demands (eg, physical activity or intercurrent febrile illness) once glycogen stores to generate glucose have been depleted. Carnitine and every enzyme and transporter involved in mitochondrial fatty acid oxidation are essential for this metabolic pathway to function. Any defect leads to the accumulation and/or deficiency of carnitine and acylcarnitines in patterns that point towards the underlying fatty acid oxidation disorder (FAOD).

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency followed by very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency are the most prevalent FAODs. Prior to their inclusion in NBS, they were significant causes of sudden unexpected death, with 19 percent of patients with unrecognized MCAD deficiency not surviving the first catabolic event triggered by common childhood illnesses such as febrile infections and gastroenteritis [20]. Some patients can present shortly after birth. Thus, follow-up for all FAODs except for carnitine uptake defect (CUD) and short-chain acyl-CoA dehydrogenase (SCAD) deficiency must be initiated immediately upon receipt of the abnormal screening result, which should be reported no later than the fifth day of life (table 1). Key features of early-onset FAODs can include poor feeding, lethargy, hepatomegaly, muscle hypotonia, and cardiomyopathy as well as hypoglycemia. Any constellation of such signs and symptoms are considered an emergency, especially when encountered in the context of an abnormal NBS result. (See "Overview of fatty acid oxidation disorders", section on 'Diagnosis'.)

Organic acidemias — Most organic acidemias are caused by enzyme defects in the mitochondrial metabolism of branched-chain (leucine, isoleucine, valine) and other amino acids. In these disorders, acylcarnitines accumulate, albeit with less specificity than for FAODs. Biotinidase deficiency requires a separate dried blood spot (DBS) punch to perform a colorimetric or fluorometric screening assay because acylcarnitines are rarely abnormal in this condition. The clinical spectrum of organic acidemias is variable. However, most can present in the newborn period with acute, life-threatening metabolic decompensations and metabolic acidosis that can be confused with sepsis. Therefore, NBS laboratories are to report relevant results no later than the fifth day of life (table 1). (See "Organic acidemias: An overview and specific defects", section on 'Diagnosis'.)

Creatine deficiency disorders — Of the three creatine deficiency disorders described (guanidinoacetate methyltransferase [GAMT] deficiency, arginine:glycine amidinotransferase [AGAT] deficiency, and X-linked creatine transporter [SLC6A8] deficiency) (table 1), only GAMT deficiency has been proposed for NBS and was added to the Recommended Uniform Screening Panel (RUSP) in early 2023 [21]. (See "Congenital disorders of creatine synthesis and transport", section on 'Diagnosis'.)

GAMT deficiency usually comes to attention after three months of life and is characterized by developmental delay, intellectual disability, impaired speech, and epilepsy. Biochemically, it is associated with elevated guanidinoacetate (GUAC) and reduced creatine and creatinine in urine and blood, markers also measurable in DBS by tandem mass spectrometry (MS/MS) along with amino acids, acylcarnitines, and succinylacetone [5,22-24]. It is already screened for in the states of Utah and New York using GUAC and creatine. Both states have identified one newborn with GAMT deficiency (1 in 540,000 and 1 in 275,000 live births, respectively) [25]. Treatment with creatine, ornithine, and sodium benzoate was started by three weeks old, and both patients have remained asymptomatic to at least four and seven months old, which is consistent with a previous report that included several patient treated earlier in infancy [26].

AGAT deficiency, which is rarer than GAMT deficiency, presents with neurodevelopmental delay and muscle weakness and has reduced GUAC. SLC6A8 deficiency has been estimated to affect nearly 3 percent of patients with neurodevelopmental disorders and is characterized by elevated excretion of creatine in urine [27]. Whether GUAC, creatine, and creatinine measurement in NBS samples could identify AGAT and SLC6A8 deficiencies has not been reported.

Galactosemias — The primary target of NBS for galactosemia is "classic" galactosemia due to (nearly) complete absence of galactose-1-phosphate uridyl transferase (GALT) activity. This disorder has the potential to cause liver failure and susceptibility to Escherichia coli sepsis in the newborn period. Cataracts, neurodevelopmental delay, and premature ovarian failure manifest later and appear not entirely preventable. Depending upon screening strategies, NBS may also identify persons with reduced GALT activity, such as those with Duarte galactosemia, who do not require treatment [28]. Galactose/lactose intake should be withheld from any newborn with an abnormal screening result for possible galactosemia until the severe forms of galactosemia have been excluded. (See "Galactosemia: Clinical features and diagnosis", section on 'Newborn screening'.)

Screening for galactosemia is performed by measurement of GALT activity and/or total galactose concentration using colorimetric or fluorometric methods. When NBS laboratories include measurement of total galactose in their screening approach, either in combination with GALT activity measurement or as the primary screening assay, three additional disorders of galactose metabolism can be identified because of elevated total galactose. Among those, only generalized UDP-galactose-4'-epimerase (GALE) deficiency can present as severely as "classic" galactosemia and requires immediate galactose/lactose restriction. "Peripheral" GALE deficiency (limited to red and white blood cells) does not necessitate treatment, while dietary galactose restriction should be implemented for patients with galactokinase (GALK) and galactose mutarotase (GALM) deficiencies to prevent the formation of cataracts.

A false positive for galactosemia may be due to severe glucose-6 phosphate dehydrogenase (G6PD) deficiency. (See 'Glucose-6-phosphate dehydrogenase (G6PD) deficiency' below.)

Lysosomal disorders — Glycogen storage disease type II (Pompe disease, acid alpha-glucosidase deficiency), mucopolysaccharidosis type I (MPS I; alpha-L-iduronidase deficiency), and mucopolysaccharidosis type II (MPS II; iduronate-2-sulfatase deficiency) are lysosomal disorders included in the RUSP as core conditions. They are identified by NBS using either MS/MS- or fluorometry-based enzyme assays. Additional lysosomal enzyme activities can be measured simultaneously using particularly the MS/MS platform, namely galactocerebrosidase (Krabbe disease), acid beta-glucosidase (Gaucher disease), alpha-galactosidase A (Fabry disease), and acid sphingomyelinase (Niemann-Pick disease types A and B). While none of them are included on the RUSP, several states already screen for all or some of these disorders. (See "Lysosomal acid alpha-glucosidase deficiency (Pompe disease, glycogen storage disease II, acid maltase deficiency)", section on 'Newborn screening' and "Mucopolysaccharidoses: Clinical features and diagnosis", section on 'Prenatal diagnosis and newborn screening' and "Krabbe disease", section on 'Newborn screening' and "Gaucher disease: Pathogenesis, clinical manifestations, and diagnosis", section on 'Newborn screening' and "Fabry disease: Clinical features and diagnosis", section on 'Diagnosis' and "Overview of Niemann-Pick disease", section on 'Diagnosis of NPD-A and NPD-B (acid sphingomyelinase deficiency)'.)

NBS assays were developed for these disorders because of the increasing availability of treatment options that provide the greatest benefit when initiated before irreversible symptoms develop. The early-onset forms of Pompe disease and Krabbe disease, in particular, benefit primarily when treatment is initiated in the first few weeks of life. A positive test for either condition requires immediate follow-up:

●Patients with infantile-onset Pompe disease may be symptomatic, with muscle hypotonia, poor feeding, and respiratory symptoms, by the time the NBS result is available. Immediate evaluation for cardiomyopathy (chest radiograph, electrocardiograph [ECG], echocardiogram, creatine kinase, lactate dehydrogenase [LDH], alanine aminotransferase [ALT], and aspartate aminotransferase [AST]) and consultation with a metabolic specialist are warranted. (See "Lysosomal acid alpha-glucosidase deficiency (Pompe disease, glycogen storage disease II, acid maltase deficiency)", section on 'Newborn screening'.)

●Krabbe disease is rarely symptomatic at birth. However, given the rapid progression of the most severe form, a hematopoietic cell transplant should be performed in patients with infantile Krabbe disease in the first four weeks of life. To achieve the best possible outcome, upon receipt of a presumptive positive NBS result for infantile Krabbe disease, providers need to immediately consult with the metabolic specialist to determine the most appropriate transplant center and either refer directly or first obtain blood for confirmatory laboratory testing. Families are counseled about the risks and expected outcomes of either transplant or supportive care at the transplant center and can consider their preferred approach while relevant confirmatory and preparatory studies are conducted in parallel [12,29]. (See "Krabbe disease", section on 'Newborn screening'.)

It should be pointed out that there is a high frequency of genotypes causing clinically irrelevant lysosomal enzyme activity reductions (often called "pseudodeficiency"). Thus, NBS for these disorders by enzyme assay has poor specificity, leading to high false-positive rates. Screening strategies have been developed and are increasingly implemented to reduce the burden on parents/caregivers and the health care system caused by false-positive results. The finding of reduced enzyme activity is followed by measurement of additional, more specific biomarkers as second-tier tests using the original NBS specimen (table 1). The test is only reported as positive if the second-tier tests are confirmatory.

Peroxisomal disorders — X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder and was the first condition added to the RUSP that has no neonatal presentation [2,30]. ALD is caused by deficiency of a transporter protein for transfer of very-long-chain fatty acids into the peroxisomes. This transporter is encoded by the adenosine triphosphate (ATP) binding cassette subfamily D member 1 (ABCD1) gene.

ALD is categorized into three phenotypes:

●Males with the childhood cerebral form of ALD typically present in the first decade of life but rarely before three years of age. This disorder has an insidious onset with nonspecific behavioral problems and, therefore, is usually not diagnosed before irreversible and progressive neurologic symptoms develop [30]. Treatment, primarily by hormone replacement and hematopoietic cell transplantation, is beneficial only if implemented before symptoms develop and because monitoring strategies exist to properly time such treatment.

●Adrenomyeloneuropathy (AMN), the most common form of ALD, typically manifests in early adulthood in males and is characterized by progressive peripheral neuropathy and impaired adrenal function. Treatment is by supportive care and glucocorticoid replacement.

●In "Addison disease only," males present as of two years of age and require glucocorticoid replacement. Aspects of AMN usually develop in adulthood.

Most carrier females present after 40 years of age with neurologic symptoms similar to AMN.

An NBS testing approach that measures lysophosphatidylcholines (LPC), particularly C₂₆-LPC, by MS/MS was developed for the childhood cerebral form of ALD (table 1). LPC are not specific to ALD and are also elevated in patients with other peroxisomal disorders, especially peroxisomal biogenesis disorders, such as Zellweger spectrum diseases that are often clinically apparent shortly after birth. Because these peroxisomal disorders have no specific treatment, they are not the target of NBS. Infants with Aicardi-Goutières syndrome may also be identified through elevated LPC [31]. (See "Autoinflammatory diseases mediated by interferon production and signaling (interferonopathies)", section on 'Aicardi-Goutières syndrome' and "Clinical features, evaluation, and diagnosis of X-linked adrenoleukodystrophy", section on 'Newborn screening'.)

While not specific, LPC are sensitive markers for ALD in males and peroxisomal biogenesis disorders. Accordingly, abnormal NBS results for elevated LPC are rarely false and, in healthy-appearing newborns, are most likely due to ALD of some form. However, NBS is unlikely to identify all newborn carrier females. In addition to the challenge of consulting families about the uncertain clinical course of their asymptomatic newborn, evaluation of family members (plasma very-long-chain fatty acid analysis and/or molecular genetic testing of ABCD1), particularly of older brothers, the mother, and maternal uncles, is advised. There is no genotype/phenotype correlation, and ALD may present differently even within a family.

An alternative screening strategy for ALD is to target males only. A four-tier approach for this purpose is under investigation in the Netherlands where NBS specimens that are positive for elevated C₂₆-LPC are tested for the number of X chromosomes. If only one X chromosome is present, C₂₆-LPC is measured again by a more specific analysis, and, if still elevated, molecular genetic analysis of the ABCD1 gene is performed. Only those cases with a variant are referred for follow-up [32].

Glucose-6-phosphate dehydrogenase (G6PD) deficiency — G6PD deficiency is a common X-linked recessive condition that can cause hemolytic anemia and neonatal jaundice, including kernicterus, when affected patients are exposed to oxidative triggers, such as specific drugs, infections, and the ingestion of fava beans (favism). Treatment is removal and lifelong avoidance of the offending agent. G6PD deficiency affords some degree of protection from malaria and, due to this evolutionary advantage, is most prevalent in ethnic populations from areas where malaria is common. Thus, NBS is performed primarily in these regions. In the United States, only Pennsylvania includes G6PD deficiency in their NBS program. However, the most commonly used NBS test for GALT deficiency (galactosemia) can inadvertently identify severe cases of G6PD deficiency because it requires active G6PD to generate nicotinamide adenine dinucleotide phosphate (NADPH) in the last step of the coupled fluorometric enzyme assay [33]. Accordingly, G6PD deficiency should be considered when galactosemia has been excluded following an abnormal NBS result. (See "Genetics and pathophysiology of glucose-6-phosphate dehydrogenase (G6PD) deficiency" and "Diagnosis and management of glucose-6-phosphate dehydrogenase (G6PD) deficiency".)

ONGOING CHALLENGES AND POSSIBLE SOLUTIONS

Performance and cost — As the number of conditions added to the NBS screening panel increases, so may the number of false positives and the cost of testing. False-positive results occur when the control and disease range of a screening marker overlap, and cutoffs are set to avoid any false-negative outcome. False-positive results for amino acid disorders are also common in (premature) neonates that require intensive care, including total parenteral nutrition. Results from a 2006 study suggested that false-positive rates in the US could be as high as 2.4 percent [34].

False-positive results can negatively impact the parent/caregiver-child relationship [35], although this can be mitigated through appropriate communication with parents/caregivers about the abnormal screening result, the likelihood that it is a true positive, and the potential implications if the newborn is affected [36,37]. Guidance on how to have these discussions with families has been developed by the Advisory Committee on Heritable Disorders in Newborns and Children (ACHDNC).

Attempts have also been made to improve the performance of screening laboratories through implementation of second-tier or reflex tests, as well as the development of bioinformatic tools to analyze the increasing number of results available for each newborn [6,38].

Second-tier tests — Second-tier screening tests have been developed, particularly as reflex tests to primary screening markers with overlapping reference ranges for affected and unaffected newborns. Typically, a primary test and cutoff are chosen to maximize sensitivity, accepting a larger number of false-positive results to avoid missing any affected patients. For newborns with a "positive" primary test, a second-tier test to measure a more specific biomarker or group of biomarkers is performed using the same NBS sample. The more specific the test is, the better it is at identifying patients with the disease. If a second-tier test is normal, it overrides the "positive" or equivocal primary screening test result, meaning the NBS result is reported as negative.

A two-tier approach was first used for NBS of tyrosinemia type I in the Canadian province of Québec, where this inborn error of tyrosine degradation is particularly common. The use of tyrosine concentrations as a primary screening marker causes a high false-positive rate because tyrosine concentrations overlap between patients with type I, as well as unaffected newborns with or without transient hypertyrosinemia. However, alpha-fetoprotein and the pathognomonic marker succinylacetone are elevated only in tyrosinemia type I. Alpha-fetoprotein analysis was used for several years as a second-tier test [39] before it was replaced by quantitative measurement of succinylacetone as the second-tier test, leading to a complete elimination of false-positive screening results [40]. Subsequently, it became possible to measure succinylacetone as part of the amino acid and acylcarnitine analysis by tandem mass spectrometry (MS/MS), and this approach is now employed by all NBS programs in the US [41]. Other second-tier tests for IEM have been applied inconsistently. Programs that do not have the ability to perform each second-tier test in-house can collaborate with other programs or clinical laboratories to provide this service [6].

Bioinformatics — One bioinformatics system, called Collaborative Laboratory Integrated Reports (CLIR; formerly Region 4 Stork [R4S]), integrates the screening test results and available demographic variables (eg, birth weight, gestational age, age at specimen collection, and sex) to determine a likelihood of disease score. This system uses data from millions of unaffected newborns as well as data from newborns who were confirmed to be affected or whose screening results turned out to be false positive [42,43]. In a retrospective review of amino acid and acylcarnitine results from approximately 175,000 California newborns, it was shown that the application of CLIR versus the routine use of analyte cutoffs would have reduced the false-positive rate by more than 10-fold and improved the positive predictive value by more than fivefold [38]. A screening program's performance can be further improved when second-tier tests are used to investigate cases that cannot be resolved by bioinformatics alone. The avoidance of false-positive results could save the health care system more than USD $1000 per case to be resolved in addition to the prevention of parent/caregiver stress and inconvenience [4].

Late-onset phenotypes of screened conditions and identification of affected relatives — When NBS began in the 1960s, the intent was to identify newborns with conditions that require immediate intervention to prevent morbidity, disability, and mortality. The assumption was that screened conditions have only one phenotype characterized by clinical onset during the first months of life. However, once population-wide screening began, milder phenotypes were quickly discovered. Other phenotypes that could have equally detrimental effects when presenting beyond the newborn period were identified through NBS but also by increased availability of biochemical genetic testing of symptomatic patients (eg, by urine organic acid analysis). Examples are variant maple syrup urine disease (MSUD) and fatty acid oxidation disorders (FAODs), such as medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, where catabolism due to common infections or other causes of stress (prolonged fasting) can elicit life-threatening metabolic decompensations [44-48]. However, phenotypes such as variant MSUD are not always detected by screening of an anabolic newborn, which emphasizes the general rule that a negative screening test should be ignored when evaluating a symptomatic patient [44,49].

When Pompe disease and mucopolysaccharidosis type I (MPS I) were added to the Recommended Uniform Screening Panel (RUSP), it was with awareness that the more common, later-onset, attenuated variants of these conditions would also be identified through screening, most likely more frequently than the classic disease variants with early onset [50]. By contrast, X-linked adrenoleukodystrophy (X-ALD) was the first condition added to the RUSP that has no neonatal phenotype, with affected males typically not presenting during the first few years of life [30]. Irrespective, X-ALD was recommended and then approved for inclusion on the RUSP in 2016 because treatment (hormone replacement and hematopoietic cell transplantation) was deemed beneficial only if implemented before symptoms develop [51], monitoring strategies were available to properly time such treatment, and a screening test was available [2]. Long-term outcome data from patients identified through prospective NBS are still needed to determine the efficacy of NBS for this disease [52].

NBS may also lead to identification of family members with the same or a related defect who are asymptomatic or minimally affected at the time of diagnosis. Mothers with carnitine deficiency, for example, are regularly identified through the screening of their unaffected newborns [53-58] Targeted screening of relatives is particularly important for late-onset conditions. As an example, when X-linked conditions such as ALD and Fabry disease are identified by NBS, testing is recommended for the mother and older siblings, especially males, and genetic counseling is recommended for the extended maternal family [52].

Adding new disease-specific tests versus large-scale DNA sequencing — The need for additional NBS will continue to expand as treatment options become available for conditions that benefit from initiation of therapy before the onset of symptoms. However, the amount of blood collected for NBS on dried blood spots (DBS) is limited. Some conditions can be added through minor modifications of existing technologies, particularly those making use of tandem mass spectrometry (MS/MS). Examples are creatine deficiency disorders, which can be screened for using the same 3 mm DBS punch along with the current disorders of amino acids, fatty acid oxidation, and organic acid metabolism [5,22,23,25,59]. Other conditions that can be combined with screening for Pompe disease and MPS I are additional lysosomal disorders and cerebrotendinous xanthomatosis (CTX) [60,61].

The limited amount of specimen available and the progress in large-scale genomic analysis has also led to the suggestion of moving NBS from biochemical to molecular genetic analysis [62]. Options include population screening limited to known pathogenic variants in genes associated with treatable conditions, sequencing of genes associated with the conditions included in NBS programs, whole exome or whole genome analysis of infants in neonatal intensive care units, and population-wide genomic sequencing [63-66].

Genomic sequencing is performed in a few NBS laboratories associated with academic centers where it is either studied under research protocols or routinely used as a second- or third-tier test to clarify abnormal primary screening test results [66-72]. One study identified a case of partial biotinidase deficiency by whole exome sequencing that was not detected by routine NBS [73], while another found that whole exome sequencing had a sensitivity of only 88 percent versus 99 percent by MS/MS to identify patients with inborn errors of amino acid, fatty acid, and organic acid metabolism and transport [74].

The cost, bioinformatic challenges, turnaround time, and ethical considerations have precluded application of genomic sequencing as a primary NBS test. A proposed pilot project, the United Kingdom Newborn Genomes Programme, aims to perform whole genome sequencing on up to 200,000 newborns [75]. Only conditions that present in childhood and have accessible and effective treatments will be reported (approximately 200 conditions compared with the 9 screened for in the existing UK NBS program). However, concerns about the challenges and ethics of this approach remain.

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Inborn errors of metabolism".)

SUMMARY AND RECOMMENDATIONS

●Overview of newborn screening – Most disorders for which newborn screening (NBS) is available are inborn errors of metabolism (IEM) and include disorders of amino acid, carbohydrate, fatty acid, and organic acid metabolism and transport. However, the conditions included on NBS panels and the actual laboratory tests and testing strategies employed by NBS laboratories vary from program to program. Except for the bedside tests for hearing loss and critical congenital cyanotic heart disease, all NBS relies on newborn blood samples collected on filter paper (dried blood spots [DBS] on Guthrie cards). (See 'Overview of NBS' above and "Overview of newborn screening".)

●Recommended Uniform Screening Panel – The US Health Resources and Services Administration established the Recommended Uniform Screening Panel (RUSP), a list of conditions that all state NBS programs should include in order to minimize variability. This list includes both core conditions that every newborn should be screened for and secondary conditions that can be detected when screening for a core condition but that do not qualify as such due to lack of effective treatment. (See 'Recommended Uniform Screening Panel (RUSP)' above.)

●Action sheets and laboratory testing algorithms for positive tests – The American College of Medical Genetics and Genomics (ACMG) Newborn Screening Translational Research Network (NBSTRN) has available on their website one-page action sheets (ACT Sheets) and laboratory testing algorithms to inform clinicians about the implications of a screening result, the urgency of follow-up, as well as the basic and specialized laboratory tests that are necessary to clarify a potential differential diagnosis or identify a false-positive screening result (table 1). (See 'ACT Sheets and algorithms' above.)

●Examples of NBS for specific IEM:

•Fatty acid oxidation disorders – Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency and very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency are the most prevalent fatty acid oxidation disorders (FAODs) and, prior to their inclusion in NBS, were significant causes of sudden unexpected death. Follow-up for most FAODs must be initiated immediately upon receipt of the abnormal screening result, which should be reported no later than the fifth day of life. Key features of early-onset FAODs can include poor feeding, lethargy, hepatomegaly, muscle hypotonia, and cardiomyopathy as well as hypoglycemia. Any constellation of such signs and symptoms are considered an emergency, especially when encountered in the context of an abnormal NBS result. (See 'Fatty acid oxidation disorders' above and "Metabolic emergencies in suspected inborn errors of metabolism: Presentation, evaluation, and management".)

•Galactosemias – The primary target of NBS for galactosemia is "classic" galactosemia due to (nearly) complete absence of galactose-1-phosphate uridyl transferase (GALT) activity. This disorder has the potential to cause liver failure and susceptibility to Escherichia coli sepsis in the newborn period. Depending upon screening strategies, NBS may also identify persons with reduced GALT activity who do not require treatment. Galactose/lactose intake should be withheld from any newborn with an abnormal screening result for possible galactosemia until the severe forms of galactosemia have been excluded. A false positive for galactosemia may be due to severe glucose-6 phosphate dehydrogenase (G6PD) deficiency. (See 'Galactosemias' above.)

•Lysosomal disorders – There is a high frequency of genotypes with clinically irrelevant lysosomal enzyme activity reductions (often called "pseudodeficiency"). Thus, NBS for these disorders by enzyme assay has poor specificity, leading to high false-positive rates. Screening strategies have been developed to reduce the burden on parents/caregivers and the health care system caused by false-positive results. When employed, the finding of reduced enzyme activity is followed by measurement of additional, more specific biomarkers as second-tier tests using the original NBS specimen. The test is only reported as positive if the second-tier tests are confirmatory. (See 'Lysosomal disorders' above.)

●Ongoing challenges and possible solutions – NBS test cutoffs are chosen to maximize sensitivity to avoid missing any affected patients. The number and cost of false-positive tests can be reduced through second-tier testing and bioinformatics. Screening can be expanded by adding disease-specific tests or possibly may be accomplished through large-scale deoxyribonucleic acid (DNA) sequencing, although concerns about the challenges and ethics of this approach remain. NBS may miss some disease variants, which emphasizes the general rule that a negative screening test should be ignored when evaluating a symptomatic patient. By contrast, NBS may also lead to identification of family members with the same or a related defect who are asymptomatic or minimally affected at the time of diagnosis. Genetic counseling is advised. (See 'Ongoing challenges and possible solutions' above.)