Neonatal Screening by Tandem Mass Spectrometry

Abstract

After completing this article, readers should be able to: The screening of newborns for inherited metabolic disorders is a well-established public health activity, first implemented in the early 1960s for the presymptomatic identification of patients who have phenylketonuria (PKU). Since then, significant technological advances, most importantly the development of tandem mass spectrometry (MS/MS), have enabled the detection of an increasing number of metabolic disorders in newborn blood. This article reviews the basic technology of MS/MS and its application to newborn screening, with the aim of highlighting the benefits and pointing out some limitations of this powerful technology.Early screening for PKU used the bacterial inhibition assay of Guthrie, in which inhibition of bacterial growth on an agar medium, introduced by the addition of a phenylalanine analog, was overcome by the presence of dried blood discs containing high levels of phenylalanine (such as from a PKU patient). This approach later was adapted for the detection of leucine in patients who have maple syrup urine disease (MSUD), methionine in patients who have classic homocystinuria, and tyrosine in patients who have tyrosinemia. Over the years, other techniques have been implemented to screen for additional disorders, including congenital hypothyroidism, biotinidase deficiency, galactosemia, hemoglobinopathies, and congenital adrenal hyperplasia. The classic criteria used to determine whether a disorder is suitable for screening include: 1) the disorder is clinically and biochemically well defined, 2) it is associated with significant morbidity or mortality, 3) an effective treatment is available, and 4) there is a simple and safe screening test. (1) By the early 1990s, all states offered screening for at least PKU and congenital hypothyroidism, with many states testing for up to seven additional disorders.MS/MS initially was applied to the analysis of newborn blood spots in 1990, with the retrospective identification of a number of metabolic disorders in archived samples of patients who had proven metabolic disease. (2) In 1997, the first large-scale, prospective study of MS/MS newborn screening was implemented in North Carolina, followed by similar studies in Massachusetts, Australia, and Germany. In particular, these studies demonstrated that the presymptomatic identification and treatment of patients who had medium-chain acyl-CoA dehydrogenase (MCAD) deficiency significantly reduced the morbidity and mortality associated with this disorder. This was seen as analogous to the screening success that had been realized with PKU and provided the impetus, in many cases responding to strong lobbying efforts of families affected with metabolic disorders, for states to modify screening protocols to include MS/MS. These studies are summarized in a number of excellent reviews.(3)(4)(5)(6)(7)It is important to realize that in the United States, the disorders contained in newborn screening panels, as well as the details of testing protocols and follow-up strategies, vary widely from state to state. As of August 2005, expanded screening for all or most disorders currently detectable by MS/MS is mandated in 28 states; an additional 14 states offer testing for an abbreviated list of disorders, including MCAD deficiency. Nine states and the District of Columbia currently do not offer MS/MS newborn screening as a state-organized activity, although testing still can be obtained from a number of commercial laboratories. It is essential for clinicians to be familiar with the specific newborn screening panels and the genetics services and resources in their home states (as well as neighboring states); an excellent resource for this is the National Newborn Screening and Genetic Resource Center (http://genes-r-us.uthscsa.edu/).MS/MS newborn screening detects two types of compounds: acylcarnitines and amino acids. Acylcarnitines are defined as any organic compound conjugated to carnitine. Carnitine itself is a small-molecular weight compound essential for the import of long-chain fatty acids into the mitochondria prior to beta-oxidation. Carnitine also serves an important detoxification role for abnormally accumulating acyl-CoA compounds, as occurs in certain organic acidemias and fatty acid oxidation disorders, via the formation of readily excreted acylcarnitine conjugates.The acylcarnitines detected by MS/MS newborn screening are carnitine conjugates derived from intermediates of fatty acid oxidation and organic acid metabolism. The fatty-acyl conjugates range in carbon length from C2 (acetylcarnitine) to C18 (octadecanoylcarnitine) as well as free, unesterified carnitine (C0). Unsaturated intermediates (eg, decenoylcarnitine [C10:1]) and hydroxylated compounds (eg, 3-hydroxy-palmitoylcarnitine [C16-OH]) also are distinguished. Organic acid intermediates forming carnitine esters include those derived from branched-chain amino acid metabolism, such as propionylcarnitine (C3) and isovalerylcarnitine (C5). A complete list of fatty acid and organic acid disorders detectable by MS/MS newborn screening is shown in Tables 1 and 2.Amino acids detected by MS/MS include aromatic amino acids (phenylalanine, tyrosine), branched-chain amino acids (leucine, isoleucine, and valine), sulfur-containing amino acids (methionine), and some intermediates of the urea cycle (citrulline, ornithine, and arginine). A complete list of amino acid disorders detectable by MS/MS screening is shown in Tables 3 and 4.The tandem mass spectrometer (Fig. 1) is a sophisticated detector consisting of five basic elements: 1) an electrospray ion source; 2) the first mass spectrometer (MS1), which filters parent compounds based on molecular weight; 3) a collision cell, which fragments compounds into smaller parts or “daughter ions”; 4) the second mass spectrometer (MS2), which filters the daughter fragments based on a specified parameter (eg, molecular weight); and 5) a detector, which detects and measures the abundance of the filtered ions.Data from the tandem mass spectrometer is analyzed using a computer algorithm that relates information gathered essentially simultaneously from MS1 and MS2. This allows for the identification of compounds based on common structural features shared between groups of molecules. Results are plotted as a histogram of ion abundance at each parent molecular weight (Fig. 2). An important consequence of this approach is that the instrument cannot differentiate between parent compounds of identical molecular weight, such as leucine and isoleucine (isomers) or isovalerylcarnitine and methylbutyrylcarnitine (both 5-carbon carnitine esters). Such discrimination requires chromatographic separation of the sample prior to introduction into the MS/MS, which is not considered necessary for the purposes of screening.MS/MS screening identifies 30 to 40 different metabolic disorders; the exact number varies from list to list because different programs tally the variant forms of the disorders differently. The combined incidence of all disorders detectable by MS/MS screening is estimated at 1 in 4,000 to 5,000, with the most commonly detected being PKU, MCAD deficiency, propionic acidemia, and methylmalonic acidemia. Other relatively commonly encountered conditions include short-chain aceyl-CoA dehydrogenase (SCAD) deficiency and 3-methylcrotonyl-CoA carboxylase (MCC) deficiency, although the clinical significance in many of these cases is not clearly understood. Still other disorders are detected at a frequency of less than 1 per 1,000,000 live births (eg, homocystinuria and multiple acyl-CoA dehydrogenase deficiency), but are still included in screening programs because they are identified in the same analysis. The following highlights some of the conditions most likely to be encountered by pediatricians in general practice.Clinically significant SCAD deficiency is a rare disorder associated with a wide spectrum of phenotypes, typically including developmental delay and muscle hypotonia. Patients are identified by screening results that include abnormally elevated C4-carnitine concentrations and elevated excretion of ethylmalonic and methylsuccinic acids on follow-up urine organic acid analysis. The diagnosis is confirmed by in vitro acylcarnitine profile (also called fatty acid oxidation flux studies) or SCAD enzyme activity assay on cultured fibroblasts. (8) Elevated C4-carnitine concentrations, however, are relatively common in newborn screening programs and, in most cases, are due to the presence of one of two common polymorphisms in the SCAD gene, 511C>T and 625G>A. These single-base changes occur in 22% and 3% of the healthy population, respectively, and by themselves do not lead to pathologically significant disease. (9) They may, however, be associated with an increased susceptibility to disease in the context of other environmental and genetic factors that are not well understood. Issues surrounding the inclusion of SCAD in newborn screening programs and the appropriate follow-up procedures following an abnormal result remain somewhat controversial and require additional studies of screening outcomes for clarification.MCAD deficiency is the most common of the fatty acid oxidation disorders and is characterized by episodes of hypoketotic hypoglycemia that can lead to lethargy, coma, brain damage, or sudden death. Onset is typically around age 2 years, although patients have been described with neonatal as well as adult presentations. (10)(11) MS/MS screening for MCAD deficiency identifies patients who have elevated C8-carnitine values; some programs also evaluate the C10:1 values and the ratio of C8 to C10:1. The diagnosis is confirmed by evaluation of the plasma acylcarnitine profile; urine organic acid and acylglycine analyses can serve as useful adjunct tests.A single MCAD mutation, 985A>G, accounts for more than 80% of MCAD alleles and is particularly common in patients of European ancestry. DNA testing for this and other MCAD mutations also may serve as useful follow-up. (12) Treatment includes avoidance of prolonged fasting, as well as maintenance of hydration and normoglycemia during periods of illness. Prior to newborn screening, approximately 20% of MCAD patients died before they were diagnosed, with a significant number of surviving patients suffering from serious neurologic sequelae. (13) With screening, the morbidity and mortality otherwise associated with this disorder has fallen to nearly zero.VLCAD deficiency is associated with three distinct phenotypes: 1) neonatal hypertrophic cardiomyopathy, hepatocellular disease, and early death; 2) onset in infancy with hypoketotic hypoglycemia and hepatic involvement; and 3) mild, later onset with myoglobinuria, muscle weakness, and myalgia. Affected patients are identified by newborn screening with elevated concentrations of long-chain acylcarnitines, in particular C14:1 (tetradeceneoylcarnitine), and the diagnosis is confirmed by plasma acylcarnitine profile. DNA studies or fatty acid oxidation flux studies in cultured fibroblasts also can be useful. Some patients identified by screening appear to be asymptomatic but presumably are at increased risk of developing symptoms later in life, although the natural history of these milder forms is not known. (14) Treatment involves the avoidance of fasting, often supplemented with medium-chain triglycerides, although the response to treatment in severe cases may be poor.LCHAD deficiency is relatively common among the fatty acid disorders, with phenotypes ranging from early-onset cardiomyopathy and death to milder, later-onset rhabdomyolysis and chronic progressive sensorimotor neuropathy and retinopathy. A single LCHAD mutation, G1528>C, accounts for more than 85% of mutations in patients who present clinically with LCHAD and appears to be associated with a more severe phenotype. Patients are identified by MS/MS screening with elevated levels of C16-OH (hydroxy-palmitoylcarnitine) and confirmed by plasma acylcarnitine profile. Fatty acid oxidation flux studies in fibroblasts also may be useful in predicting the degree of disease severity. (15) A related disorder, mitochondrial trifunctional protein deficiency, is associated with identical biochemical abnormalities and can be differentiated by specific enzyme assays. Treatment includes avoidance of fasting, often with reduction of dietary long-chain fats and supplementation of medium-chain fats and L-carnitine. Although this approach reduces the incidence of neurologic injury associated with metabolic attacks, it does not eliminate the risk of recurrent rhabdomyolysis and retinopathy later in life.Primary carnitine deficiency arises from defects in carnitine transport across the plasma membrane and in the renal tubule. This results in extremely low carnitine levels in plasma, muscle, and other tissues and abnormally elevated urinary excretion of carnitine. Clinical findings range from neonatal hypertrophic cardiomyopathy to later-onset muscle weakness and cardiomyopathy. Some patients also experience recurrent Reye-like episodes of hypoketotic hypoglycemia. Patients who have carnitine uptake deficiency are identified by newborn screening with abnormally low levels of free carnitine (C0); the diagnosis is confirmed by evaluation of plasma and urine carnitine levels as well as functional studies of the carnitine transporter in cultured fibroblasts. Because heterozygous parents of affected individuals also may have impaired carnitine transport and be at risk of disease, (16) they should be evaluated as well. Treatment is by supplementation with high doses of L-carnitine.PA results from a deficiency of propionyl-CoA carboxylase, a biotin-requiring enzyme in the catabolic pathway of valine and isoleucine. MMA arises from a block in the next step in the pathway that is mediated by methylmalonyl-CoA mutase (mutase), an enzyme that requires the cofactor adenosylcobalamin (Ado-B12). MMA is a heterogeneous group of disorders arising from either a deficiency of mutase itself or from defects in the synthesis of Ado-B12. The classic, severe presentation of either PA or MMA is a neonatal life-threatening illness associated with secondary hyperammonemia and anion gap metabolic acidosis. Patients who have later-onset disease present with recurrent episodes of metabolic decompensation, developmental delay, and mental retardation. Both PA and MMA patients are identified by screening with elevated levels of C3 (propionylcarnitine); the elevation of C4DC (methylmalonylcarnitine) in MMA patients is often not apparent. The diagnosis is clarified by urine organic acid analysis. Additional follow-up studies include propionyl-CoA carboxylase assay (to confirm PA) or complementation studies in cultured fibroblasts to (to delineate the specific form of MMA). Long-term treatment includes dietary restriction of PA and MMA precursors, most importantly valine and isoleucine, and supplementation with L-carnitine. Some patients who have MMA also respond to supplementation with cobalamin. The prognosis in severe cases still may be poor despite aggressive treatment.GAI arises from a deficiency of glutaryl-CoA dehydrogenase in the degradative pathways of lysine and tryptophan. Affected patients typically present between 3 and 18 months after birth with acute, irreversible dystonia and basal ganglia damage, often triggered by an intercurrent illness. Screening identifies elevated levels of C5DC (glutarylcarnitine), and the diagnosis is confirmed by urine organic acid analysis and quantitative glutaric and 3-hdyroxyglutaric acid levels. Variant GAI patients have been described who excrete only minimally elevated levels of glutarate metabolites. (17) For these patients, specific enzyme assays or DNA studies may be needed to confirm the diagnosis. Treatment involves avoidance of fasting and supplementation with L-carnitine; some centers also treat with dietary restriction of lysine and tryptophan. Presymptomatic treatment has been shown to prevent the onset of disabling dystonia in the majority of patients. IVA results from the deficiency of isovaleryl-CoA dehydrogenase activity in the degradative pathway of leucine. The presentation and range of clinical phenotypes is similar to that for PA and MMA, including severe, early-onset, and milder forms. Affected patients often are described as having an odor reminiscent of sweaty feet. Newborn screening identifies patients who have elevated levels of C5 (isovalerylcarnitine), the same biochemical marker seen in patients who have 2-methylbutyryl-CoA dehydrogenase deficiency. These two disorders are easily distinguished by urine organic acid and acylglycine analyses. Treatment of IVA is by dietary restriction of leucine and supplementation with glycine and L-carnitine. MCC is a biotin-requiring enzyme in the catabolism of leucine. MCC deficiency is associated with clinical phenotypes ranging from severe and life-threatening to apparently benign, but patients typically present in infancy with poor feeding, lethargy, and hypotonia together with hypoglycemia and metabolic acidosis. Although once thought to be very rare, this condition now is being detected at an unexpectedly high frequency in newborn screening programs. Affected patients are identified with abnormally elevated C5-OH (3-hydroxyisovalerylcarnitine); this marker also identifies patients who have hydroxymethylglutaryl-CoA lyase deficiency, beta-ketothiolase deficiency, and 3-methylglutaconic aciduria. These disorders all can be distinguished by urine organic acid analysis. Elevated C5-OH also has been seen as a transient finding in newborns as well as in biotinidase deficiency and in infants of mothers who have asymptomatic MCC deficiency. (18) Therefore, in some cases, appropriate follow-up also may include the evaluation of the acylcarnitine profile and urine organic acids in the mother as well as serum biotinidase activity in the patient. Treatment of MCC deficiency is by avoidance of fasting and catabolism as well as carnitine supplementation.PKU results from the deficiency of phenylalanine hydroxylase (PAH), a biopterin-requiring enzyme that catalyzes the conversion of phenylalanine to tyrosine. Classic PKU is defined as an untreated blood phenylalanine concentration greater than 20 mg/dL (1,210 mcmol/L), with non-PKU hyperphenylalaninemia variants characterized by phenylalanine levels of less than 20 mg/dL (1,210 mcmol/L) (normal levels, 0.5 to 1.5 mg/dL [30 to 91 mcmol/L]). Hyperphenylalaninemia also arises from defects in the synthesis or recycling of the biopterin cofactor required for proper enzyme activity. Patients who have PKU or its variants are identified by MS/MS screening with an elevated level of phenylalanine and ratio of phenylalanine to tyrosine. The diagnosis is confirmed by quantitative amino acid analysis and biopterin cofactor studies; DNA testing for PAH mutations is becoming more widespread and may help to delineate patients who respond to biopterin therapy. (19) Conventional treatment is by dietary restriction of phenylalanine, which prevents the otherwise profound mental retardation seen in untreated patients.Tyrosinemia type I (fumarylacetoacetate hydrolase deficiency) is associated with hepatomegaly and acute liver failure that presents in infancy and is characterized by the abnormal accumulation of the metabolite succinylacetone. Type II (tyrosine aminotransferase deficiency) and type III (4-hydroxyphenylpyruvate dioxygenase deficiency) do not involve hepatic dysfunction, but are characterized by variable mental retardation and, in the case of type II, hyperkeratosis of the palms and soles. Many states do not include tyrosinemia in their screening panels because of the relatively high false-positive rate that results from transient tyrosinemia, a benign condition that is particularly common in preterm infants. For those states that do screen for tyrosinemia, the second-tier identification of succinylacetone can help to lower the false-positive rate and identify patients who have tyrosinemia type I, (20) the most clinically serious of the disorders. Type I patients are treated with NTBC, a compound that inhibits the production of succinylacetone, as well as a diet low in phenylalanine and tyrosine. (21)MSUD results from the deficiency of branched-chain alpha-ketoacid dehydrogenase (BCKAD), an enzyme common to the pathways of valine, isoleucine, and leucine. The classic, severe presentation of MSUD involves life-threatening neonatal illness accompanied by ketoacidosis, respiratory distress, and coma. Milder forms may present with episodes of ketoacidosis and neurologic impairment, with progressive mental retardation and spasticity. MS/MS screening identifies MSUD patients who have elevated levels of leucine and isoleucine. Because these analytes are not separated during the analysis, results are reported as their sum. The most definitive follow-up test is plasma amino acid analysis; urine organic acids and BCKAD assays also often are performed. Long-term treatment is by dietary restriction of branched-chain amino acids, with some patients also responding to thiamin.AS deficiency (citrullinemia) and AL deficiency (argininosuccinicaciduria) are urea cycle disorders that have similar clinical presentations, most frequently involving profound neonatal hyperammonemia, lethargy, coma, and early death. Both disorders are identified by MS/MS by abnormally elevated citrulline concentrations, with some programs also detecting argininosuccinic acid in AL patients. The diagnosis of either disorder is confirmed by plasma amino acid analysis, which identifies abnormally elevated citrulline values in both disorders as well as elevated argininosuccinic acid in AL. Long-term treatment is by use of ammonia-scavenging medications (sodium benzoate and phenylbutyrate) and dietary protein restriction, (22) although the prognosis in severe disease may remain poor.CBS is a vitamin B6-requiring enzyme in the metabolic pathway of methionine. Clinical findings of CBS deficiency include variable mental retardation, ectopia lentis, skeletal abnormalities, and increased risk for thromboembolism and early stroke. Although elevated homocystine excretion is the hallmark of this disorder, screening identifies patients who have abnormal elevations of blood methionine, the upstream precursor of homocystine. Elevated methionine concentrations also are seen in patients who have methionine adenosyltransferase deficiency, an apparently benign condition associated with persistent, isolated hypermethioninemia. It also can result from prematurity, liver disease, and certain diets. Screening does not identify patients who have other forms of homocystinuria, including disorders of cobalamin or folate metabolism (eg, methylenetetrahydrofolate reductase deficiency), which are associated with low levels of methionine. CBS deficiency is confirmed by studies of plasma amino acids and total homocystine as well as CBS enzyme assay. Treatment is by dietary restriction of methionine, with some patients also responding to pyridoxine therapy.The overall false-positive rate for MS/MS screening is about 0.1% or 1 in 1,000 live births in laboratories throughout the country, although the actual false-positive rate varies from laboratory to laboratory and even from analyte to analyte. Among the many underlying explanations for a false-positive result are a cutoff value very close to the normal range, prematurity, liver dysfunction, diet, medication, and maternal nutrition status. Some results are “true positives” in that they reflect a real biochemical abnormality that is not believed to be associated with clinically significant disease. Examples of this include mildly elevated C5-OH-caritine, which often diminishes to normal levels over time; elevated C4-carnitine associated with benign polymorphisms of the SCAD gene; and mildly elevated C8-carnitine in variants of MCAD deficiency.On the other hand, a number of cases of true metabolic disease have been missed by MS/MS newborn screening, including infants who have GAI, cobalamin variants of methylmalonic acidemia, argininosuccinicaciduria, and LCHAD deficiency. Although false-negative results rarely occur because of clerical or laboratory errors, the more common explanation is that the analyte in question was only minimally elevated, as can occur in milder variants forms of the disorders, and did not exceed the screening threshold. These cases underscore the need for ongoing clinical vigilance, even in the face of a normal newborn screening result, and the recognition that diagnostic testing should be pursued whenever clinically indicated, regardless of prior screening.Even with the extraordinary benefits derived from MS/MS newborn screening, it is important to acknowledge some limitations inherent in current screening practices. Many disorders are not currently detected by MS/MS screening, including several of the urea cycle disorders (ornithine transcarbamoylase deficiency and carbamoylphosphate synthetase I deficiency), disorders of pyruvate metabolism and oxidative phosphorylation (including pyruvate dehydrogenase deficiency and the mitochondrial respiratory chain defects), lysosomal storage disorders, and disorders of metal metabolism (Wilson disease, molybdenum cofactor deficiency). In addition, the screening efficacy for some disorders included in panels as “potentially detectable” is not known, as with nonketotic hyperglycinemia (glycine encephalopathy), malonic aciduria, and CPT-I deficiency. Therefore, it is important to recognize that a negative result, even on an “expanded” screen of metabolic disorders, does not rule out the possibility of a metabolic disease.A second caveat is that for some disorders, especially those associated with an acute, neonatal presentation, the benefits of early detection and treatment are not clear. Concerns have been raised about long-term outcomes of patients who have urea cycle defects, propionic acidemia, and methylmalonic acidemia. For some patients, early intervention does not appear to alter the natural history of disease. (23)(24) This raises ethical issues because MS/MS screening may identify some patients who have diseases that currently are untreatable, at apparent odds with the original criteria that once defined a “screenable” disorder.Finally, many, if not all, metabolic disorders encompass a wide clinical spectrum that includes mild, or even benign, variant forms. Although screening may identify some of these variant cases by virtue of biochemical abnormalities, the clinical significance of these findings and their natural history in the absence of treatment often is not known. For example, the number of cases of MCAD deficiency identified by newborn screening far exceeds the number presenting clinically, (25) and many of these patients harbor an MCAD mutation that has only subtle effects on enzyme structure and function. (26) Therefore, the question of whether or how to treat patients who have mild biochemical abnormalities identified by screening is not clear and requires additional studies on the relationships between specific molecular mutations, protein function, and clinical outcome.Additional disorders currently are being evaluated for their feasibility as MS/MS screening targets, including the lysosomal storage disorders (eg, Fabry, Gaucher and Pompe diseases) (27) and peroxisomal disorders (X-linked adrenoleukodystrophy). (28)(29) Other disorders being considered as potential screening candidates using other methodologies include Wilson disease, (30) fragile X syndrome, and congenital hearing loss. (31) The motivation behind much of this development lies in the increasing availability of new treatment strategies, such as bone marrow transplantation, enzyme replacement therapy, and specific pharmaceutical agents, which often show maximum efficacy when implemented as early in the disease course as possible. In the meantime, MS/MS represents a powerful technology that, when applied to the screening of newborns for metabolic disorders, has had a profound impact on our ability to identify patients and implement early, lifesaving treatments. It is clear that this field is still rapidly growing and will continue to evolve in the years ahead.