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Review of Newborn Screeningn the Era of Tandem Mass Spectrometry:

hat’s New for the Pediatric Neurologist?ara Copeland, MD

Newborn screening has evolved from a single test for a single metabolite to a test thatdetects more than 90 metabolites on a single blood spot. In the past decade, the panel ofnewborn-screening disorders has rapidly expanded and will continue to grow as more isdiscovered about the human genome. It continues to be a very sensitive populationscreening tool that is susceptible to the status of the infant and the timing of the specimencollection. This review discusses the disorders that should be detected on neonatalbloodspot screening and what pediatric neurologists may see in those that were detectedon newborn screening and treated and those that have been untreated.Semin Pediatr Neurol 15:110–116 © 2008 Elsevier Inc. All rights reserved.

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ir Archibald Garrod first used the term “inborn errors ofmetabolism” in 1908 to describe alkaptonuria. Since the

nitial description, over 1,000 inherited disorders of intermedi-ry metabolism have been described. In the 1960s, newborncreening (NBS) via a bloodspot was introduced as a way toetect and start early treatment for phenylketonuria to preventhe neurodevelopmental problems found in untreated patients.n the 1970s, the ability to test for congenital hypothyroidismllowed another addition to the neonatal blood spot panel. Sincehen, various disorders were added to state NBS panels, oneisorder at a time. Each disorder required the development ofew testing materials, and each test was run separately.1

In 1990, the first report of using tandem mass spectrome-ry (TMS) on blood spots was published.2 This technologyllowed for the testing of multiple acylcarnitines and aminocids on a single blood spot to be run in a matter of minutesmultiplex testing). The metabolites detected could be corre-ated with various disease states. Where previously one bloodpot could detect one disorder, it is now possible to detectpwards of 80 disorders on a single blood spot. Within 8ears of the original article being published, several states hadmplemented this new technology and were screening for

ultiple disorders shortly after birth.1 In 2005, the Americanollege of Medical Genetics (ACMG) set out to develop rec-mmendations for a standard NBS panel designed to ensure

rom the University of Iowa Children’s Hospital, Iowa City, IA.ddress reprint requests to Sara Copeland, MD, University of Iowa Chil-

dren’s Hospital, 200 Hawkins Drive W133GH, Iowa City, IA 52242.

gE-mail: sara-copeland@uiowa.edu

10 1071-9091/08/$-see front matter © 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.spen.2008.05.003

ll newborns, no matter where they were born, had the besthance for a good outcome. The ACMG published a consen-us statement in 2006 that recommended a core panel of 29isorders and 25 additional disorders that will also be pickedp in the core panel screen (secondary targets). The recom-endation was that these should be screened for in every

tate.3 As of the end of 2007, more than 45 states are screen-ng for the recommended core panel, and in the next year oro all 50 states will be screening for the recommended panel.4

isorders Not Screened on TMSntil the late 1990s, each test for the NBS panel was per-

ormed separately. The majority of screening is now per-ormed as a multiplex test in which multiple analytes areeasured at the same time. There is still a group of otherisorders that are not covered by TMS. These include theemoglobinopathies, endocrine disorders, and enzymatic as-ays for galactosemia and biotinidase deficiency. These areutlined in Table 1. Table 2 outlines the various features thatay be observed by pediatric neurologists in these patients.he screening for these disorders is not new, and, although

hey have implications for the neurologist, they will not beiscussed in depth in this article apart from Table 2.

isorders Covered by TMShere are several categories of disorders covered in the ACMG-ecommended core panel, including amino acids disorders, or-

anic acidurias, urea cycle defects, and mitochondrial fatty-acid

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Tandem mass spectrometry 111

xidation defects. The sensitivity for the disorders varies de-ending on the timing of the screen, the metabolic status of the

nfant, the quality of the laboratory, and the interpretation ofesults. Overall, the sensitivity is close to 99% for the core dis-rders, and most states have about a 0.02% false-positive rate,gain depending on the status of the infant and timing of thepecimen collection. As a screening test, it is not diagnostic andhould not be assumed to confirm the absence or presence of theisease. Thus, infants presenting with symptoms consistentith a newborn-screened disorder who had a normal neonatal

creen still need to be further evaluated.5 The core panel ofisorders is outlined in Table 3, and secondary targets are listed

n Table 4.3

isorders ofmino Acid Metabolism

MS can measure the amount of various amino acids presentn the blood spot. The amino acids on the panel were chosenor their ability to indicate the presence of specific disorders.n general, they should be detectable after 24 hours of ageespite feeding status and, when disease is present, will con-inue to increase with time. External factors can cause falselevations including hyperalimentation and liver dysfunction.he presentation of primary disorders of amino acid metabolism

n general is insidious and slow, and patients do not commonlyave major metabolic decompensations. However, when un-reated, the symptoms may present to neurologists as ataxia,ental retardation, and seizures. Treatment consists of limiting

he offending amino acid in the diet and, in the case of tyrosine-ia type I, blockade of the proximal enzyme step. See Table 5

or an outline of neurologic findings.The following are what pediatric neurologists may see in pa-

ients with disorders of amino acid metabolism: (1) magneticesonance imaging white-matter changes in untreated patients,

able 1 Disorders Screened for in the Neonatal Period not viaMS

Core Disorder Category of Disease

ickle cell disease Hemoglobinopathyeta-thalassemia HemoglobinopathyC disease Hemoglobinopathyongenital hypothyroidism Endocrinopathyongenital adrenal hyperplasia Endocrinopathyiotinidase deficiency Enzyme defect, organic

acidurialassic galactosemia Enzyme defect, sugar

intoleranceearing screen Congenital hearing loss

Secondary targets Category of disease

ariant hemoglobinopathies Hemoglobinopathyalatokinase deficiency, GALK Variant enzyme defect of

galactose metabolismpimerase deficiency, GALE Variant enzyme defect of

galactose metabolism

2) neuropathy when untreated, and (3) ataxia when untreated.

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isorders of the Urea Cyclehere are 6 well-categorized disorders of urea cycle metabo-

ism. Newborn screening will only detect 3 of the enzymeefects. Classic citrullinemia and argininosuccinic aciduriare the typical urea cycle disorders. The third enzyme in thisathway presents as an aminoacidopathy, argininemia. Ureaycle defects present secondary to the inability to metabolizexcess nitrogen to urea and the body builds up ammonia,hich causes lethargy and vomiting and can proceed to

oma. Long-term outcomes depend on how elevated the am-onia level is and how long it remains elevated.6 The body

ttempts to regulate the ammonia formation by shunting ex-ess nitrogen to form glutamine, which acts as an osmoticgent, causing astrocyte swelling and can cause permanentrain damage and seizures. If the enzyme defect is severenough, even when detected and appropriately managedhrough newborn screening, the infants may not survive orave a good outcome.7 Treatment for urea cycle disorders isrotein restriction, supplementation of missing amino acids,nd nitrogen-scavenging medications.

All urea cycle disorders have late-onset variants that ifissed on a neonatal screen can present with developmentalelay, liver dysfunction, ataxia, and seizures. The patientsay have a history of recurrent bouts of emesis associatedith illness or increased protein intake.6 Even when treatedrospectively, patients will have metabolic decompensations

able 3 Core Panel in ACMG Guidelines for TMS Disordersnd Their Categories

Core Disorder Category of Disease

omocystinuria Aminoacidopathyyrosinemia type I Aminoacidopathyhenylketonuria Aminoacidopathyrgininosuccinic aciduria Urea cycle defectitrullinemia type I Urea cycle defectaple syrup urine disease Organic aciduria

sovaleric aciduria Organic acidurialutaric aciduria type I Organic aciduriaethylmalonic aciduria Organic aciduriaropionic aciduria Organic aciduria-hydroxy-3-methylglutaryl-CoA Lyase deficiency

Organic aciduria

ultiple carboxylasedeficiency

Organic aciduria

eta-ketothiolase deficiency Organic aciduriaedium-chain acyl-CoAdehydrogenase deficiency

Beta-oxidation defect

ery long-chain acyl-CoAdehydrogenase deficiency

Beta-oxidation defect

ong-chain L-3-hydroxyacyl-CoA dehydrogenasedeficiency

Beta-oxidation defect

rifunctional proteindeficiency

Beta-oxidation defect

arnitine-uptake defect Beta-oxidation defect-carnitine transporter defect

elated to illness despite being compliant with therapy. Ulti- D

ately, the ammonia peak and the duration of the ammonialevation determines the final outcome.8 See Table 5 for anutline of neurologic findings.The following are some of the symptoms that pediatric neu-

ologists may encounter in patients with urea cycle defects: (1)ncephalopathy and mental status changes, (2) cerebral edemarom high glutamine levels that may lead to brainstem hernia-ion if untreated, (3) developmental delay is common evenhen well treated, and (4) basal ganglia injury can occur anday be associated with movement disorders after recovery.

rganic Acidurias/Acidemiass the category name would suggest, this group of disordersauses metabolic acidosis and is often associated with a largenion gap. Most organic acidemias result from dysfunction ofspecific step in amino acid catabolism. This then causes thexcretion of nonamino organic acids in urine. The majority ofhe classic organic acid disorders result from dysfunction in

able 4 Secondary Targets for TMS Disorders and Their Cat-gories

Secondary Targets Category of Disease

ild hyperphenyalaninemia Aminoacidopathyyrosinemia type II Aminoacidopathyiopterin cofactor defects Aminoacidopathyrgininemia Aminoacidopathyyrosinemia type III Aminoacidopathyypermethioninemia Aminoacidopathyitrullinemia type II–citrin defect Aminoacidopathyethylmalonic aciduria withhomocystinuria (Cbl C, D)

Organic aciduria

sobutyryl-CoA dehydrogenasedeficiency (IBD)

Organic aciduria

-methyl 3-hydroxy butyricaciduria (2MHBD)

Organic aciduria

-Methylbutyryl-CoAdehydrogenase deficiency

Organic aciduria

-Methylglutaconic aciduria Organic aciduriahort chain acyl-CoAdehydrogenase deficiency

Beta-oxidation defect

lutaric aciduria type II ormultiple acyl CoAdehydrogenase deficiency

Beta-oxidation defect

edium-/short-chain L-3-hydroxyacyl-CoA dehydrogenasedeficiency

Beta-oxidation defect

edium-chain ketoacyl-CoAThiolase deficiency

Beta-oxidation defect

arnitine palmitoyltransferase IIdeficiency

Beta-oxidationdefect- carnitinetransporter defect

arnitine:acylcarnitinetranslocase deficiency

Beta-oxidationdefect- carnitinetransporter defect

arnitine palmitoyltransferasedeficiency type I

Beta-oxidationdefect- carnitinetransporter defect

ienoyl-CoA reductase deficiency Beta-oxidation defect

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Tandem mass spectrometry 113

he branched-chain amino acid metabolism or the break-own of lysine. These patients are generally well at birth, andhe goal is to identify them before the onset of symptoms viaeonatal screening. The standard of care for these disordershen detected on NBS is to start therapy before 10 days of

ge. The longer the delay in starting treatment is, the worsehe long-term outcome. Clinical presentation is generallyoxic encephalopathy and includes vomiting, poor feeding,eurologic symptoms such as seizures and abnormal tone,nd lethargy progressing to coma. Milder variants that are notiagnosed on NBS may present in the older child or adoles-ent as loss of intellectual function, ataxia or other focal neu-ologic signs, Reye syndrome, recurrent keto-acidosis, orsychiatric symptoms. A variety of magnetic resonance im-ging abnormalities have been described in the organic aci-emias, including distinctive basal ganglia lesions in glutaricciduria type I, white-matter changes in maple syrup urineisease, and abnormalities of the globus pallidus in methyl-alonic acidemia. Treatment is dietary restriction of the pre-

ursor amino acid, vitamin supplementation of cofactors,nd rapid treatment during periods of illness to limit long-erm complications.9 See Table 6 for an outline of neurologicndings.The following are symptoms found in organic acidurias

hat may be seen by pediatric neurologists: (1) seizures oftenfter metabolic decompensation, particularly if associatedith mental status changes; (2) basal ganglia strokes andovement disorders; (3) magnetic resonance imaging

hanges may occur if chronically untreated or after acuteecompensation; and (4) developmental delay or specific de-elopmental/learning problems, even when well treated.

eta-Oxidationefects: Carnitineransporter Defects Causingatty-Acid Oxidation Defects

n order for long chain fatty acids to be metabolized to acetyl-oA and eventually to adenosine triphosphate, they requireransport into the mitochondria. There are 4 enzymes that areesponsible for this transport; 3 are involved in the transportf the fatty acids using translocations of carnitine and acyl-oA. These include carnitine palmitoyl transferase type I,arnitine:acylcarnitine translocase, and carnitine palmitoylransferase type II. They have some difference on the acylcar-itine profile for the neonatal screen, but presentation is veryimilar with hypoketotic hypoglycemia, cardiomyopathy,habdomyolysis, and liver dysfunction. Presentation reflectshe deficit in energy production when the body is forced toely on fatty-acid oxidation for energy. The fourth enzymenvolved in fatty-acid transport is the high-affinity carnitineptake transporter that is responsible for recycling carnitine

n the muscle, heart, and kidneys. Defects in this recyclingechanism cause primary carnitine deficiency. This disorder

s called carnitine uptake deficiency. The presentation of car-

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Table 6 Neurologic Findings in Organic Aciduria Disorders14

Disorder MRI Changes Size AtaxiaDevelopmental

DelayHearing

Loss Neurop OtherAbnormal

Tone Stroke

Isovaleric aciduria U U U U- hemorrhageGlutaric aciduria type I U/T: neuronal loss and fibrous

gliosis in the caudate andputamen

U/T U/T U/T U/T basal gangliastroke withdecompensate

Methylmalonic aciduria U: hyperintensity in globuspallidus T2

U U U U U/T basal gangliastroke withdecompensate

Maple syrup urine disease U: hyperintense signal whitematter; periventricular deepwhite matter and subcorticalU-fibers; globus pallidus,thalami, and brainstem.

U U U U U/T basal gangliastroke withdecompensate

Propionic aciduria U/T: increase signal intensityglobus pallidus

U U U U U/T: basalganglia strokewithdecompensate

3-hydroxy-3-methylglutaryl-CoA Lyase deficiency(HMG coA lyase)

U: diffuse mild signal intensitycerebral white matter

U U U U U: retinopathy U U: hypoglycemia

Isobutyryl-CoAdehydrogenasedeficiency (IBD)

2-methyl 3-hydroxy butyricaciduria (2MHBD)

U/T: frontotemporal atrophyand bilateral signalabnormalities in theputamen

U/T U/T U/T U/T U/T: Movementdisorder;retinal degen.

U/T

Multiple carboxylasedeficiency

U:18 subependymal cysts U U U U: opticatrophy;alopecia

U

Beta-ketothiolasedeficiency

U: increased T2 intensitywithin the posterior lateralpart of the putamen

U

Methylmalonic aciduriawith homocystinuria (CblC, D)

U/T:21 demyelinating process U/T U/T U/T U/T U/T: lenssublux;retinopathy.

U/T U/T: thromboticor metabolic

U � found mostly in untreated patients; T � treated patients may have symptoms.

114S.Copeland

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Tandem mass spectrometry 115

orter defects.10 See Table 7 for an outline of neurologicndings.The following is a summary of fatty-acid oxidation defects

nd how they may present to pediatric neurologists: (1) sei-ures are common sequelae to hypoglycemic episode, (2)agnetic resonance imaging changes may be found afteretabolic decompensation, and (3) developmental delay andsychiatric problems are long-term consequences to recur-ent hypoglycemia events.

isorders ofitochondrial Beta-Oxidation

fter fatty acids enter the mitochondria, they are brokenown into acetyl-CoA for entry into the mitochondrial aden-sine triphosphate complex and energy formation. Eight de-ects in beta-oxidation are detectable via TMS on newbornlood spots (see Tables 3 and 4). Each enzyme is responsibleor fatty acids of specific chain lengths and hydroxylation.here is some overlap between the enzymes, but the markersre fairly specific to the various disorders. All beta-oxidationefects can potentially result in hypoketotic hypoglycemia.11

owever, the defects that occur in long-chain fatty-acid me-abolism tend to present with more liver and cardiac prob-ems because those organs have higher energy requirementsnd rely on fatty-acid oxidation as a major source of energy.he medium-chain defects predominantly present with hy-oglycemia, arrhythmias, and sudden death. The short-chainefects may or may not be medically significant.14 See table 7

or an outline of neurologic findings.The following is a summary of fatty-acid oxidation defects

nd how they may present to pediatric neurologists: (1) sei-ures are common sequelae to a hypoglycemic episode, (2)agnetic resonance imaging changes may be found afteretabolic decompensation, and (3) developmental delay andsychiatric problems are long-term consequences to recur-ent hypoglycemia events.

onclusioneurologists should be aware that NBS, although fairly sen-

itive, does have some limitations, and they need to be atten-ive to the common symptoms that may indicate a metabolicisorder. At this time, we are screening for about 60 meta-olic disorders on the neonatal blood spot, but that leavesore than 900 other disorders that are not detected on NBS.

o, although a normal NBS is reassuring, it does not rule outmetabolic defect.With the introduction of TMS and the idea of multiplex

able 7 Neurologic Findings in Fatty-Acid Oxidation Defects:

Disorder MRI Changes

atty acid oxidationdefects

U: cerebral occipital and parietal regiochanges from hypoglycemia

� found mostly in untreated patients; T � treated patients may h

esting (multiple disorders tested at one time), the number of

ossible disorders that may be included for NBS have in-reased exponentially. At one time, each test was time inten-ive and required separate interpretation, but this is not thease anymore.

As the technology improves and new therapies evolve, theisorders that various programs are investigating as possibledditions to their NBS panel continues to grow. Keeping upo date with the state panel will be an ongoing challenge.

For example, although no one is screening for Duchenneuscular dystrophy in a population-wide manner, there is a

ot of interest in the newborn-screening community andmong the various groups interested in this group of disor-ers. There are also several groups with a strong interest tocreen for Fragile X syndrome.

New challenges will become evident as new disorders aredded to the NBS panel. The responsibilities of the subspe-ialist may move away from initial diagnosis to chronic man-gement of disorders. Diseases will be diagnosed muchooner than was ever thought possible. The expectations forhese patients and their long-term outcomes will also changes we begin screening, detecting, and treating before acuteecompensations can have a chance to cause death or dis-bility.

In the future, we may have a “neurology” panel that canetect mutations in multiple genes associated with seizuresr loss of skills like CNS glucose transporter deficiency, neu-otransmitter defects, creatine transporter defects, or pyri-oxine-dependent seizures. The era of genomics is openingoors, and the possibilities are endless. There will always beneed for pediatric neurologists, but with genomic screeninge may be looking at a new way of managing patients—morereventative and prospective care than evaluation and diag-osis of patients with unknown disorders.

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