Newborn Screening for SevereCombined Immunodeficiency and T-cellLymphopenia in California, 2010–2017George S. Amatuni, BS,a,b Robert J. Currier, PhD,a Joseph A. Church, MD,c Tracey Bishop,d Elena Grimbacher,e

Alan Anh-Chuong Nguyen, MD,f Rajni Agarwal-Hashmi, MD,g Constantino P. Aznar, PhD,d Manish J. Butte, MD, PhD,h

Morton J. Cowan, MD,a Morna J. Dorsey, MD, MMSc,a Christopher C. Dvorak, MD,a Neena Kapoor, MD,c Donald B. Kohn, MD,h

M. Louise Markert, MD, PhD,i Theodore B. Moore, MD,h Stanley J. Naides, MD,j Stanley Sciortino, PhD, MPH,d

Lisa Feuchtbaum, DrPH, MPH,d Rasoul A. Koupaei, PhD,d Jennifer M. Puck, MDa

abstractOBJECTIVES:Newborn screening for severe combined immunodeficiency (SCID) was instituted inCalifornia in 2010. In the ensuing 6.5 years, 3 252 156 infants in the state had DNA from driedblood spots assayed for T-cell receptor excision circles (TRECs). Abnormal TREC resultswere followed-up with liquid blood testing for T-cell abnormalities. We report theperformance of the SCID screening program and the outcomes of infants who were identified.

METHODS: Data that were reviewed and analyzed included demographics, nursery summaries,TREC and lymphocyte flow-cytometry values, and available follow-up, including clinicaland genetic diagnoses, treatments, and outcomes.

RESULTS: Infants with clinically significant T-cell lymphopenia (TCL) were successfully identifiedat a rate of 1 in 15 300 births. Of these, 50 cases of SCID, or 1 in 65 000 births (95%confidence interval 1 in 51 000–1 in 90 000) were found. Prompt treatment led to 94%survival. Infants with non-SCID TCL were also identified, diagnosed and managed, including4 with complete DiGeorge syndrome who received thymus transplants. Although no cases oftypical SCID are known to have been missed, 2 infants with delayed-onset leaky SCID hadnormal neonatal TREC screens but came to clinical attention at 7 and 23 months of age.

CONCLUSIONS: Population-based TREC testing, although unable to detect immune defects in whichT cells are present at birth, is effective for identifying SCID and clinically important TCL withhigh sensitivity and specificity. The experience in California supports the rapid, widespreadadoption of SCID newborn screening.

WHAT’S KNOWN ON THIS SUBJECT: Earlier resultsshowed that newborn screening can be highly effectivefor the early detection of severe combinedimmunodeficiency (SCID).

WHAT THIS STUDY ADDS: Screening 3.25 millionCalifornia infants of diverse ethnicity with the T-cellreceptor excision circle test substantiates the value ofnewborn screening for the diagnosis of SCID and non-SCID T-cell lymphopenia. Both are more common andgenetically heterogeneous than previously thought.

To cite: Amatuni GS, Currier RJ, Church JA, et al. NewbornScreening for Severe Combined Immunodeficiency andT-cell Lymphopenia in California, 2010–2017. Pediatrics.2019;143(2):e20182300

aDepartment of Pediatrics, University of California, San Francisco and Benioff Children’s Hospital, San Francisco,California; bDepartment of Cell Biology, Stem Cell Institute, Albert Einstein College of Medicine, Bronx, New York;cDepartment of Pediatrics, Keck School of Medicine, University of Southern California and Children’s Hospital LosAngeles, Los Angeles, California; dGenetic Disease Screening Program, California Department of Public Health,Richmond, California; eSchool of Architecture and Urban Planning, University of Stuttgart, Stuttgart, Germany;fBroward Health Medical Center, Fort Lauderdale, Florida; gDepartment of Pediatrics, School of Medicine, StanfordUniversity, Palo Alto, California; hDepartment of Pediatrics, University of California, Los Angeles and University ofCalifornia, Los Angeles Mattel Children’s Hospital, Los Angeles, California; iDepartment of Pediatrics, School ofMedicine, Duke University, Durham, North Carolina; and jImmunology Department, Quest Diagnostics NicholsInstitute, San Juan Capistrano, California

Dr Puck conceptualized and designed the study, collected and analyzed data, and revised the

manuscript; Mr Amatuni collected and analyzed data and wrote the initial manuscript; Dr Currier

collaborated on the study design, conducted data analysis and interpretation, and critically

reviewed and revised the manuscript; Drs Church, Cowan, Dorsey, Dvorak, Agarwal-Hashmi, Butte,

Kapoor, Kohn, Markert, and Moore contributed essential clinical data and reviewed and revised the

manuscript; Ms Bishop and Drs Aznar, Feuchtbaum, Sciortino, and Koupaei organized (Continued)

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Severe combined immunodeficiency(SCID) encompasses ∼20 known (andadditional unknown), rare geneticdisorders of adaptive immunity.Individuals who are affected lack Tlymphocytes and have absent ornonfunctional B lymphocytes,rendering them susceptible to life-threatening bacterial, fungal, and viralinfections with both ordinary andopportunistic pathogens.1,2 Because,20% of case patients with SCIDhave a recognized positive familyhistory and because infants who areaffected appear healthy at birth,population-based newborn screening(NBS) is the only strategy to identifyall infants with SCID sufficiently earlyto provide treatment before the onsetof infectious complications.3–7

Treatment of SCID most oftenrequires an allogeneic hematopoieticcell transplant (HCT) from a suitablymatched healthy donor; in addition,enzyme replacement therapy (ERT) isavailable for adenosine deaminase(ADA)–deficient SCID, and autologoushematopoietic cell correction by genetherapy (GT) has been successfulfor ADA-deficient and X-linkedSCID.1,2,7–10 Best outcomes areachieved if infants are treated early toavoid infectious complications, asdocumented in single-center reportsand by multicenter studies from thePrimary Immune DeficiencyTreatment Consortium (PIDTC).2,4,7,11

The PIDTC has establishedlaboratory-based definitions for SCIDindependent of complications, such asopportunistic infections or failure tothrive.12 Infants with typical SCIDhave ,300 autologous T cells per mL,absent naïve T cells, and proliferativeresponses to the mitogenphytohemagglutinin that are ,10%of control values. They often havespontaneous engraftment of maternalT cells, and the diagnosis is furtherconfirmed by documentingpathogenic mutations in known SCIDgenes. In addition, some infants havehypomorphic mutations in SCIDgenes that allow residual function

and produce a leaky phenotype.These infants usually have ,1500autologous T cells per mL unless therehas been oligoclonal proliferation ofmemory phenotype T cells, as occursin Omenn syndrome; infants withleaky SCID generally have sufficientT-cell function to prevent maternalengraftment, but immunity isinsufficient to fight infections.

The timely presymptomatic diagnosisof SCID is the primary goal of SCIDNBS. California instituted population-wide screening for SCID in August2010, following pilot programs inWisconsin, Massachusetts, and theNavajo Reservation and on therecommendation by the AdvisoryCommittee on Heritable Disorders inNewborns and Children (endorsed bythe Secretary of the Department ofHealth and Human Services) thatscreening for SCID be added to theRecommended Uniform ScreeningPanel.13–16 The SCID NBS test isbased on detection of T-cell receptorexcision circles (TRECs), DNAbiomarkers of normal Tlymphopoiesis that can be measuredby a polymerase chain reaction testby using dried blood spots (DBSs)routinely obtained from newborns byheel stick.3 DBSs from newborns withSCID have undetectable or lownumbers of TRECs. Moreover, a lowTREC copy number in a DBS is usedto detect not only the primaryscreening target, SCID, but alsoseveral non-SCID conditionscharacterized by T-cell lymphopenia(TCL) that may also benefit fromearly diagnosis and intervention toprevent infections.

In 2014, researchers of an analysis ofearly pooled data from 11 screeningprograms endorsed the effectivenessof TREC screening.17 Although TREC-based SCID screening was highlyeffective for the diagnosis of SCID inall participating programs, the lack ofuniformity in assay methodologybetween the programs precludeddetermination of rates of recall forfurther testing. As of December 10,

2018, all 50 states in the UnitedStates, plus the District of Columbiaand Puerto Rico, as well as anincreasing number of countries,including Israel, New Zealand, andNorway, have adopted universal NBSfor SCID. Additional regional or pilotprograms are under way in manyother countries. Here we report theexperience and outcomes of 6.5 yearsof screening .3.25 million newbornsfor SCID in California, the state withthe largest number of births and highethnic diversity.

METHODS

Details of methods, including thestatistical analysis, are in theSupplemental Information. Briefly,DBS specimens collected fromessentially all infants born inCalifornia since mid-2010 had TRECtesting for SCID, initially witha laboratory-developed assay at theGenetic Disease Laboratory (GDL) inRichmond, California, as published.18

The EnLite neonatal TREC kit(PerkinElmer, Inc, Waltham, MA) wasadopted in June 2015, after whichtesting moved to regional NBSlaboratories. Reported TREC andb-actin copy numbers differedbetween these methods, requiringadjustment of thresholds. Thecategories of test results aresummarized in Table 1. After 1positive or 2 incomplete TREC testresults, infants had liquid bloodanalyzed for lymphocyte subsets andnaïve and memory T-cell markers ata single laboratory (QuestDiagnostics, San Juan Capistrano, CA).

Lymphocyte phenotypes wereinterpreted by California Departmentof Public Health (CDPH) immunologyassociates (M.J.B., J.A.C., S.A.M., andJ.M.P.). Infants with ,300 CD3 T cellsper mL of blood or with ,2% of theirhelper T cells bearing the naïve T-cellmarker CD45RA were referred topediatric hospitals with experience intreating SCID (Children’s Hospital LosAngeles; Stanford Lucille Packard

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Children’s Hospital; University ofCalifornia, Los Angeles MattelChildren’s Hospital; or University ofCalifornia, San Francisco BenioffChildren’s Hospital). Additionally,infants with up to 1500 circulatingT cells per mL with naïve T cellspresent were referred to pediatricimmunology clinics for furthermanagement. Long-term follow-upwas requested from transplantcenters and immunologists accordingto the standard, comprehensive CDPHprogram to monitor outcomes of allCalifornia infants with positive NBSresults.19 Children with SCID or TCLdetected by NBS were managed untilresolution or for at least 1 year andup to 7.5 years. Diagnoses wereestablished by standard clinicaltesting, including gene sequencing,except as noted below.

RESULTS

Between August 15, 2010, and March31, 2017, a total of 3 252 156newborns had SCID screening. Ofthese, 562 (1 of 5800 total births) hadnonnormal TREC results followed byliquid blood lymphocyte phenotypingby flow cytometry (Fig 1A). Althoughonly 9% of all infants were in NICUs,this population had over half of theTREC-positive screening results(53%; n = 298; Fig 1B). Cases weretracked to establish diagnoses of boththe primary target of screening, SCID,and non-SCID TCL of ,1500 T cellsper mL, a clinically importantsecondary target. This T-cell cutoffwas chosen to be highly selective but

to avoid exposing infants withsignificant immune compromise tothe otherwise-mandated live,attenuated rotavirus vaccine, whichcan cause serious diarrhea inimmunodeficiency.20 TCL wasdocumented in 38% (n = 213) of thecohort of infants requiring follow-upflow-cytometry tests or in 1 per15 300 total newborns (95%confidence interval [CI] 1 per13 500–1 per 17 700). An urgentpositive TREC result (Table 1) wasa specific predictor of both SCID andnon-SCID TCL; of the 82 newbornswith urgent positive results, 38(46%) had SCID and 21 (26%) hadnon-SCID TCL. Positive or incompleteTREC test results and documentedTCL in those infants who hadabnormal TREC test results weremore common in infants born veryprematurely, as assessed bygestational age (GA) at birth or bybirth weight (Tables 2 and 3; seeSupplemental Information for furtherdetails). Ethnicity and sex were notsignificant risk factors for havinga positive or incomplete TREC resultor TCL (data not shown). TRECscreening identified 50 cases of SCID(1 per 65 000 births [95% CI 1 per51 000–1 per 90 000]), 44 cases fromregular nurseries and 6 cases fromNICUs. The proportions of casepatients with true SCID in NICUs wasthe same as in regular nurseries (Fig 1B and C). The 162 cases of non-SCIDTCL were categorized as congenitalsyndromes (72 cases), includingmultisystem congenital abnormalitieswith TCL and non-SCID single-gene

primary immunodeficiencies; as TCLresulting from another condition andresolving once that condition wascorrected (25 cases); as preterm birthalone, in which TCL was self-limited ifthe infant survived (33 cases); or asidiopathic TCL (33 cases).

Cases of SCID

Of the 50 infants with animmunophenotype of SCID, 49 wereimmediately hospitalized at animmunodeficiency center fortreatment with allogeneic HCT,autologous corrected cell GT, or ERT(Table 4). One infant with SCID bornto a foreign visitor left the UnitedStates before receiving any furtherimmune evaluation or treatment. Allinfants with SCID were healthy atdiagnosis except for 2 who hadalready been brought to medicalattention with infection or rashes and1 who was born prematurely and hadfeeding intolerance, bronchiolitis, andleukopenia.21 Only 2 were known byproviders at birth hospitals to be atrisk for SCID because of a positivefamily history, whereas 7 others whowere identified by SCID NBS hadolder relatives who were affected anddiagnosed with SCID, and 1 hada sibling who had died of infantilepneumonia consistent with SCID. Themedian age at flow cytometry forinfants lacking a recognized familyhistory of SCID was 21 days (range6–40 days).

Of the 49 patients who wereevaluable and treated (Table 4), 46survived (94%). Of the 3 deaths, 1

TABLE 1 TREC Test Results, Interpretation, and Actions Required

TREC Copy No., per mL b-Actin Copy No., per mL TREC Test Interpretation Action Required

.18a N/Ab Normal No further actionUndetectable or ,4c .35d Urgent positive Immediate callback for liquid blood for lymphocyte subsets4–18, infant not in NICU .35 Positive Callback for liquid blood for lymphocyte subsets4–18, infant in NICU N/A Incomplete Second DBS test in 2 wk or at discharge from nursery; after 2 incomplete

test results, liquid blood for lymphocyte subsets

N/A, not applicable.a TREC copies per mL of peripheral blood equivalent for the EnLite kit; .25 copies per mL for the original laboratory-developed test; values for both methods after adjustments based oninitial experience.b b-actin was not reported if the initial TREC value was normal.c Fewer than 6 copies per mL for the laboratory-developed test.d For the EnLite kit, the cutoff for the laboratory-developed test was .5000 copies per mL.

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infant of unknown genotype died ofcytomegalovirus (CMV) infection withencephalitis. One with recombinase-activating gene 1 (RAG1)–deficientSCID and 1 with X-linked SCID, eachof whom had received pretransplant

conditioning with high-dose busulfanchemotherapy, developed fatalhepatic sinusoidal obstructionsyndrome post-HCT; 1 of these alsohad a concomitant CMV infection thathad been present before HCT.

Table 4 indicates genotype, nurserytype, TREC result, and whether theSCID was typical or leaky. Whereas∼90% of infants with typical SCIDhad urgent positive TREC results, halfof the infants with leaky SCID hadTREC numbers in the positive, but noturgent, range. See SupplementalTable 11 for patient mutations. Asreported previously in the literature,the X-linked interleukin-2 receptor gchain (IL2RG) gene was mutated mostfrequently.1,2,7,17,18 The 9 infants withADA-deficient SCID had, in addition tolymphopenia of T, B, and naturalkiller (NK) cells, fewer neutrophils(mean 986 cells per mL) andmonocytes (mean 372 cells per mL)than the other infants with SCID, whohad means of 4700 neutrophils permL and 1600 monocytes per mL, orhealthy infants. Interestingly, thebirth weights of infants with ADA-deficient SCID (median 2608 g) alsotended to be lower than those ofother infants with SCID (median3288 g), perhaps reflecting that ADAdeficiency is a systemic disorder ofpurine salvage. Despite clinicalsequencing of known SCID genes, 5infants (10%) remained withouta proven genotype. Whole-exomesequencing failed to solve 4 of these,whereas confirmatory experimentsrevealed 1 infant to have a defect ina novel SCID gene, B cell lymphoma11B (BCL11B).22

In the right columns of Table 4outcomes after therapy for patientswith SCID are summarized. GT,consisting of an autologous transplantof bone marrow–derivedhematopoietic stem cells transducedwith a vector encoding the correctcomplementary DNA, was used to

FIGURE 1Summary of NBS for SCID in California. A, Number of infants at each stage of screening. B, Numbersand proportions of infants in regular versus intensive care nurseries at each stage of the screeningprocess. C, Final diagnosis category for infants with TCL in each nursery type. a Total infantsscreened included 91% of infants who were cared for in a regular well-infant nursery or born athome, and 9% who were cared for in a NICU. b Infants (47% from regular nurseries and 53% fromNICUs) with a liquid blood sample tested after 1 positive or 2 incomplete TREC test results made up0.17% of all births, excluding 20 newborns with TREC-positive test results who either died or werelost to follow-up before a liquid blood sample could be obtained and 2 infants born in another statewho had positive TREC screen results and relocated to California for follow-up. c Infants with ,1500T cells per mL or ,2% naïve CD3 CD4 CD45RA T helper cells (46% in the regular nursery and 54% inthe NICU) made up 0.007% of the population (1 per 15 000 births).

TABLE 2 Preterm Birth and TCL in Patients WithAbnormal TREC Test Results by GA

GA Group, wk NotTCL, n

TCL, n

22–31 114 4732–41 225 171

x2 (degrees of freedom = 1; N = 557) = 9.40; P , .05.

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treat 4 infants with X-linked SCID and8 infants with ADA-deficient SCID,with uniform survival at 2.5 to6.5 years and notable success. Allinfants receiving GT for ADA-deficientSCID have had multilineage immunereconstitution. Three recipients ofGT for X-linked SCID have recoveredT-cell immunity; 1 recipient hadfailed his initial haploidenticalT-cell-depleted allogeneic HCT, and1 recipient no longer requiredimmunoglobulin G (IgG) infusions.However, 1 recipient of GT forX-linked SCID failed this initialtreatment but recovered T- and B-cellimmunity after an allogeneictransplant from a matched,unrelated donor.

Deficiency of the recombinaseactivating proteins RAG1 or RAG2,both required for T- andB-lymphocyte antigen receptorrearrangement, caused 4 cases oftypical SCID and 7 cases of leakySCID, of which 3 patients had Omennsyndrome, in which a limited poolof poorly regulated T cells expands,causing rashes, adenopathy,splenomegaly, eosinophilia, andelevated immunoglobulin E. Anexample was an infant with leakyRAG1-deficient SCID who had .4500T cells per mL. Although this was inthe normal range, only 46 of his 3680CD4 helper T cells (1.2%) had thenaïve CD45RA phenotype.

During the 6.5 years of screening, 2cases of SCID were discovered to havebeen missed. Both had normalnewborn DBS TREC numbers, eachwell above the designated cutoff forfollow-up testing. One infant, whopresented at age 7 months withPneumocystis pneumonia, had theknown, recurrent, hypomorphic

X-linked IL2RG mutation,Arg222Cys.23 His delayed-onset leakySCID was successfully treated witha conditioned HCT from a matched,unrelated donor. The other wasa delayed-onset case of incompleteADA deficiency, diagnosed at23 months of age after repeated otitisand hospitalization for pneumonia.24

This child was successfully treatedwith GT.

Non-SCID TCL

After ruling out SCID, infants withnon-SCID TCL identified by SCID NBSwere tracked by participatingimmunologists in the CDPH long-termfollow-up program.19 Although manyinfants received a diagnosis in theneonatal period, 22 required furthertesting over the course of 3 to18 months to establish a diagnosis(Fig 2), and 33 others remainedidiopathic (Fig 2). The most frequentnon-SCID TCL conditions werecongenital syndromes (Fig 2,Table 5). Six infants with syndromesdied before flow cytometry could beperformed (Supplemental Table 12).

See the Supplemental Information fordetails of patients with non-SCID TCL.The most common syndrome wasDiGeorge syndrome, with 47 cases(Fig 2, Table 5). More than 90% hadpartial DiGeorge syndrome, in whichT cells rose to age-adjusted referenceranges12 over time or becameadequate as judged by the localimmunologist. However, 4 infantswith complete DiGeorge syndrome(having persistently ,300 T cells permL and/or absent naïve helperT cells) received thymus transplantsat Duke University in Durham, NorthCarolina, with 3 surviving for at least2 years.25–27 Trisomy 21 accounted

for 8 (11%) infants with syndromes;TCL resolved in 3 infants, 3 infantsdied of nonimmune complications,and 2 were lost to follow-up. All 5patients with ataxia telangiectasia(AT) were healthy, term newbornsfrom regular nurseries with 800 to1323 T cells per mL. Risinga-fetoprotein levels after age6 months led to the identificationof deleterious mutations in the ATmutated gene.28 One infant with ATdeveloped infections requiring IgGinfusions during his first year. Threeinfants with coloboma of the eye,heart defect, atresia choanae,restricted growth and development,genital abnormality, and earabnormality (CHARGE) syndromerequired NICU care for congenitalmalformations unrelated to theimmune system. One was consideredfor a thymus transplant but died ofrespiratory failure. The 3 casepatients with diabetic embryopathywith TCL were from NICUs and hadadditional congenital abnormalities.Single patients with rare conditionsconstituted the remainder of casepatients with syndromes (Table 5;discussed in the SupplementalInformation), including an infant witha novel short-limbed immuno-skeletaldysplasia in whom whole-exomesequencing revealed exostosinlikeglycosyltransferase 3 (EXTL3)deficiency.29

Secondary TCL in 25 case patients(Table 6) resolved to normal onceexcess T-cell losses or suppression ofT-cell maturation had been reversed.T-cell losses were associated withvascular leakage, whereas 2 infantshad poor T-cell production because ofin utero exposure to maternalimmunosuppressive medications,fingolimod in 1 and azathioprine inthe other. One infant had a thymicteratoma, with T cells normalizingafter it was resected. TCL was alsooccasionally associated with pretermbirth (Table 7, Supplemental Tables 9and 10), affecting infants born at 32weeks’ gestation or earlier (median

TABLE 3 Preterm Birth and TCL in Patients With Abnormal TREC Test Results by Birth Weight

Birth wt Group, g NotTCL, n

TCL, n

0–1500 108 42.1501 231 176

x2 (degrees of freedom = 1; N = 557) = 10.69; P , .05.

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TABLE4Characteristics,Treatm

ents,and

Outcom

esof

49InfantsWith

SCID

Diagnosedby

UsingNB

S

SCID

Genotype

(No.

Cases)

SCID

Phenotypea

Nursery

TREC

Result

Outcom

eAfterTreatm

entb

Urgent

Positivec

Positive

Died

(Posttransplant

d,Cause)

FullTandBCellRecovery

andOffIgG,

Treatm

entd

TbutNotBCells

Reconstituted,S

tillon

IgG,

Treatm

ent

IL2RG(14)

Typical

Regular

120

1(218

d,SOSe,CMV)

2cond

MUD

,1GT,1

cond

MUD

afterGT

6MMRD

,1cond

MUD

f ,1GT,1

GTafter

MMRD

NICU

20

ADA(9)

Typical

Regular

52

08GT,1

MSD

0NICU

10

Leaky

Regular

01

RAG1

(8)

Typical

Regular

20

1(28d,SOS)

1cond

MSD

0Leaky

Regular

2leaky,1Om

enn

syndrome

1leaky,2Om

enn

syndrome

04cond

MMRD

2cond

MMRD

(1of

whom

planning

tostop

IgG)

IL7R

(6)

Typical

Regular

50

04MMRD

,1MUD

,1cond

MSD

afterMSD

0Typical

NICU

01

JAK3

(3)

Typical

Regular

30

01cond

MSD,1

cond

MUD

1MMRD

RAG2

(3)

Typical

Regular

20

01MSD,2

cond

MUD

0Leaky

Regular

01

BCL11B

(1)

Leaky

Regular

10

01cond

MUD

0RM

RP(1)

Leaky

Regular

01

01cond

MUD

0Unknow

n(4)

Typical

Regular

11

1(69d,

CMV)

01cond

MUD

planning

tostop

IgG

NICU

10

00

1Cond

MUD

Leaky

Regular

10

00

1cond

MUD

planning

tostop

IgG

Cond,conditioning

with

busulfanchem

otherapy

and,in

someinstances,fludarabineor

otheragents;M

MRD

,mismatched

relateddonor;MSD,m

atched

siblingdonor;MUD

,matched

adultor

cord

bloodunrelateddonor.

aCriteriafrom

thePIDTCfortypicalSCID

andleakySCID;O

mennsyndrome,asubset

ofleakySCID,includeserythrodermarash,adenopathy,oligoclonalT-cellexpansion,

eosinophilia,andelevated

immunoglobinElevels.

bOneinfant

notincluded

inthistablehadurgent-positive

TRECsandtheT2

B2NK

+SCID

lymphocyteprofilebutlefttheUnitedStates

before

HCT;no

genotype

orfollow-upisavailable.

cThreeor

fewer

TREC

copies

with

anorm

alcontrolpolymerasechainreactioncopy

numberby

theEnLite

kitassayor

equivalent

with

aprevious

in-laboratory

assay.

dTreatm

entsincluded

GTwith

low-dosebusulfanfollowed

byautologous

CD34

+bone

marrowcells

transduced

with

acorrectcopy

ofthemutated

gene

(IL2RGor

ADA),M

MRD

(usuallyaparent

who

ishaploidentical),MSD,M

UD.Cond,conditioning

with

busulfanchem

otherapy

and(insomeinstances)

fludarabineor

otheragents

(other

infantsmay

have

hadserotherapywith

rabbitantithymocyteglobulin

oranti-CD52).

ePosttransplant

hepatic

sinusoidal

obstructionsyndromeafterbusulfanchem

otherapy.

fOnepatient

lefttheUnitedStates

afterconditioned

MUD

HCTandreconstitution;

itisnotknow

nifthepatient

isstill

onIgG.

56 AMATUNI et al by guest on July 1, 2020www.aappublications.org/newsDownloaded from

GA at birth: 24 weeks), with a medianbirth weight of 610 g (range:300–1720 g). TCL resolved or wasimproving in 26 of 33 preterm infantswho were retested.

Idiopathic TCL included cases forwhich an underlying condition couldnot be determined, even afterimmunologic evaluation and, in manyinstances, sequencing of gene panelsor whole exomes. Of the 33 cases of

idiopathic TCL (Table 8), 13 resolvedor were improving, whereas 13patients had persistent TCL, and 6patients were lost to follow-up.

DISCUSSION

TREC screening for SCID has beenused to establish the feasibility andutility of DNA-based, high-throughputscreening, which, like tandem massspectrometry, has already proven

adaptable to additional conditions(including spinal muscular atrophy, asapproved in July 2018) for addition tothe Recommended Uniform ScreeningPanel on the basis of a review ofevidence.30 TREC screening inCalifornia facilitated the diagnosis ofSCID in 50 infants over a 6.5-yearperiod, permitting timely referral tospecialized treatment centers forimmune-restoring therapy. Sensitivityappears complete in that no casesof typical or early-onset leaky SCIDwith low T cells are known to havebeen missed. Indeed, cases of SCIDwere readily identified by low TRECs,even when maternally engraftedT cells were present, or in Omennsyndrome, when up to severalthousand T cells per micro liter hadarisen from florid expansion ofa small oligoclonal T-cell pool. TRECsare generated in newly differentiatedT cells and are not replicated whenT cells undergo mitosis. Thus, bothmaternally transferred cells andproliferating autologous cells havelost TRECs through dilution, and suchcells also lack the naïve CD45RAmarker and instead express theCD45RO memory maker, both part ofthe standard California SCID flow-cytometry panel. A positive familyhistory of SCID might be expected tomake NBS redundant in at least somecase patients,5 but in our experience,obtaining such a history provedproblematic. Two of the 50 infantswith SCID were from known at-riskpregnancies and had a prenataldiagnosis. However, neonatalmedical providers were unaware that6 other infants had a previous siblingwho had been treated for SCID,including 2 siblings who themselveshad been identified by SCID NBS inCalifornia and received transplants.Although the rate of false-positiveTREC screen results was higher inNICUs than in regular nurseries(Fig 1), 6 of the 50 case patientswith true SCID (12%) were in NICUs,proportional to the total numberof infants in NICUs (9% of allnewborns).

FIGURE 2Conditions diagnosed in infants with TCL identified by NBS, including 50 infants with typical or leakySCID (including Omenn syndrome), 72 infants with a final diagnosis of a syndrome associated withT-cell impairment or of a non-SCID primary immunodeficiency disorder, 25 infants with congenitaldefects associated with secondary TCL, 33 infants with TCL and preterm birth alone, and 55 infantswith an initial diagnosis of idiopathic TCL (in whom a syndrome was later diagnosed in 22, leaving 33idiopathic cases with no underlying defect identified). a Includes 3 cases of CHARGE syndrome, 3cases of diabetic embryopathy, and 1 each of CLOVES syndrome, EXTL3 deficiency (immune-skeletaldysplasia with neurodevelopmental abnormalities), Fryns syndrome (multiple anomalies with di-aphragmatic defects), Nijmegen breakage syndrome (defect in the DNA repair protein nibrin,causing immune defects, microcephaly, short stature, and malignancy), Noonan syndrome (con-genital heart disease, characteristic facial and physical features, and short stature), and RAC2deficiency (a non-SCID primary immunodeficiency affecting neutrophils and lymphocytes). b Includes2 cases of maternal immunosuppressive medication (azathioprine in 1, fingolimod in the other) and1 case of neonatal teratoma of the thymus. c Infants with TCL associated with preterm birth alonewere grouped by birth weight.

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Survival of patients with SCID was94% at 1 to 8 years of age.Furthermore, 31 of the 46 survivingchildren (67%) have fullyreconstituted B- as well as T-cell

immunity, no longer requiring IgGsupplementation (Table 1); IgGproduction is expected to recover inadditional patients over time, raisingthe proportion of children fully cured

to 75%. The optimal treatment ofSCID, HCT from a matched sibling,was available to only 3 infants. Aspreviously published, the childrenwith X-linked SCID who received HCT

TABLE 5 Diagnoses and Mortality in 72 Infants With TCL Found by Using SCID NBS Who Had Syndromes and Non-SCID Primary Immunodeficiency, IncludingCases That Were Initially Classified as Idiopathic TCL

Syndrome (No.Cases)

Typical Clinical Characteristics Nursery Type TREC Result Outcome

Regular NICU UrgentPositive

Positive Immune Therapy(If Known)

Died DuringFollow-up, Cause

DiGeorgesyndrome (47)

Multiple congenital anomalies, including variable cardiacoutflow tract defects, parathyroid hypoplasia, thymichypoplasia, and immunodeficiency, and other congenitaldefects; most often due to interstitial deletion ofchromosome 22q11.2

19 28 3 44 4 receivedthymus

transplants

1, post–thymustransplant medical

complication

Trisomy 21 (8) Wide range of neurodevelopmental and physical defects,including hypotonia, short stature, congenital heartdisease, and defects in host defense against infection

1 7 0 8 — 3

AT (5) Cerebellar ataxia with progressive neuromusculardeterioration, telangiectasias, immune defects, andpredisposition to malignancy; DNA repair defect

5 0 0 5 1 received IgGinfusions

0

CHARGEsyndrome (3)

Ocular coloboma, heart defect, atresia choanae, restrictedgrowth and development, genital abnormality, and earabnormality

0 3 3 0 1 received IgGinfusions

1, respiratory failure

Diabeticembryopathy(3)

Multiple congenital malformations involving brain,cardiovascular, skeletal, and immune systems

0 3 0 3 TCL resolved in 1 1

CLOVESsyndrome (1)

Congenital lipomas, overgrowth, vascular malformations,epidermal nevi, and skeletal anomalies

0 1 1 0 IgG infusions 1, respiratory failure

EXTL3 deficiency(1)

Immuno-skeletal dysplasia with neurodevelopmentalabnormalities

0 1 0 1 IgG infusions 0

Fryns syndrome(1)

Multiple congenital anomalies with diaphragmatic defectsand lung maldevelopment

0 1 0 1 — 1, respiratory failure

Nijmegensyndrome (1)

Nibrin mutation causing DNA repair defect withimmunodeficiency, microcephaly, short stature, andfrequent malignancy

0 1 0 1 IgG infusions 0

Noonansyndrome (1)

Several known genotypes causing congenital heart disease,characteristic facial and physical features, and shortstature

0 1 0 1 — 1

RAC2 deficiency(1)

Immunodeficiency affecting neutrophils and lymphocytes 1 0 0 1 — 0

—, not applicable.

TABLE 6 Causes of Secondary TCL Found by using SCID NBS With TRECs

Primary Condition (No. Cases) Nursery Type TREC Result Comments (Deaths Occurred During the NICU Stay)

Regular NICU UrgentPositive

Positive

Congenital heart disease (6) 0 6 1 5 All had urgent heart surgery; 1 diedHydrops (6) 0 6 1 5 1 diedGastroschisis (4) 0 4 0 4 All had urgent surgical interventionChylothorax (2) 0 2 0 2 1 diedMaternal immunosuppressivemedication (2)

2 0 1 1 Azathioprine or fingolimod used by mothers during pregnancy; infants’ T-cell countsnormalized spontaneously

Third-space fluid leakage (2) 0 2 1 1 1 diedIntestinal atresia (1) 0 1 1 0 Urgent surgical interventionMeconium ileus (1) 0 1 1 0 Surgical intervention (ileostomy)Teratoma of the thymus (1) 0 1 0 1 Surgery; patient also had hydrops, ascites, and chylothorax

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from a parent who was haploidenticalwithout chemotherapy achievedT-cell reconstitution but continueto require IgG infusions.1,2,7,11

Chemotherapy conditioning, used inmost cases of SCID in which B-cellrecovery occurred, nonetheless didnot ensure the development ofnormal antibody production and wasassociated with 2 deaths (see below).Moreover, B-cell recovery wasachieved with unconditioned orconditioned HCT for all 6 casesof interleukin-7 receptor(IL7R)–deficient SCID, in whichB cells are intrinsically functionalonce helper T cells are restored.31,32

Autologous GT after low-dosebusulfan conditioning is available inclinical trials for patients withADA-deficient and X-linked SCID wholack matched sibling donors.8–10

All 8 recipients of GT who wereADA-deficient have had full immunerecovery, including independencefrom IgG infusions, as did the solerecipient of a matched-sibling HCTfor ADA-SCID. ERT was used asa bridging therapy between diagnosisand GT for most ADA cases. Four of14 case patients with X-linked SCIDalso received GT, although 1 requireda subsequent allogeneic HCT for

inadequate T-cell recovery.Importantly, and in contrast toprevious unconditioned GT orunconditioned HCT with donors otherthan matched siblings, current GT forboth ADA-deficient and X-linked SCID,when preceded by low-dose busulfanchemotherapy, restored both T- andB-cell immunity, allowing 10 of the 12recipients of GT to discontinue IgGinfusions.

The 3 deaths among infants with SCIDpresent a challenge to furtherimprove current therapy. One deathfollowed hepatic sinusoidalobstruction from high-dosechemotherapy, 1 was due to CMVinfection, and 1 was due to combinedCMV infection and chemotherapytoxicity. Individual targeting ofbusulfan exposure to account forvariable metabolism in young infantsmay prevent future chemotherapy-associated deaths.33 CMV is highlypathogenic in the absence of T-cellimmunity. Infants with SCID areunlikely to have congenital CMVbecause maternal primary infectionduring pregnancy is uncommon;however, they may acquire CMV at orafter birth from maternal virus shedinto secretions or breast milk becausemost mothers have serologic

evidence of previous CMV exposure.34

One infant with SCID of unknowngenotype may have acquired CMVfrom breast milk from his motherwho was CMV seropositive. Onadmission to the hospital at 21 daysof age, this infant had CMV that wasuncontrolled despite antiviral therapyand an accelerated haploidenticalpaternal HCT. California may havereduced CMV exposure time amonginfants with SCID by moving theTREC assay to regional laboratorieswith a faster turnaround. Afterregionalization, the median age atdiagnosis of SCID by lymphocytesubsets for infants withouta recognized family history was8 days (n = 5), compared with21 days (n = 40) before that time.Further study will be required toestablish the sources and bestpreventive measures to combat CMVinfection.

SCID did not occur more frequently inany ethnic group, nor did anyCalifornia population subgroupsdemonstrate predominant foundermutations, as previously identified forNavajo American Indians and theAmish.35,36 However, the frequentoccurrence of SCID because ofhomozygous autosomal recessivemutations reveals a pattern of sharedparental ancestry. Of 30 cases ofautosomal recessive SCID withmutations identified, 9 (30%) werehomozygous (SupplementalTable 11).

A late diagnosis in 2 infants who hadnormal TREC NBS but who proved tohave missense defects in the ADAand IL2RG genes, respectively, revealsthat SCID has a broad spectrumof mutations and phenotypicpresentations. Mutations that aresufficiently leaky to produce someT cells and TRECs may evadeneonatal detection. Importantly,raising the TREC cutoff would nothave helped to identify either of thesecase patients by NBS because bothhad TREC counts well above thedesignated level requiring follow-up.

TABLE 7 TCL Associated With Preterm Birth and Low Birth Weight

Birth wt, g No. Patients TREC Result

Urgent Positive Positive

300–450 5 1 4451–550 7 0 7551–650 11 1 10651–900 8 1 7901–1720 2 0 2

TABLE 8 Idiopathic TCL Detected by TREC Screening

Status at Last Follow-up (No. Cases) Nursery Type TREC Result

Regular NICU UrgentPositive

Positive

TCL resolved (11) 9 2 0 11TCL improving (2) 2 0 2 0TCL persistent (13) 12 1 3 10Infant died of respiratory failure, had a low CD4 T cell count (1) 0 1 0 1Status unknown; lost to follow-up (6) 5 1 1 5

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In addition to diagnosing SCID, TRECNBS has identified non-SCID TCL,permitting intervention to avoid liverotavirus vaccination, which cancause severe diarrheal disease ininfants with insufficient T-cellimmunity.20 Secondary andpremature TCL cases are expected toresolve, but until they do so, patientsare immunocompromised. Althoughthe TCL definition of 1500 CD3 cellsper mL as the upper limit providesa medically prudent indication tointervene with immune supervisionand avoidance of live viral vaccines,this cutoff does not ensure theidentification of all cases of non-SCID,persistent, mild TCL.

Most of the non-SCID syndromeswere multisystem disorderssummarized in Table 5; of allindividuals with these syndromes,those with low NBS TRECs and,1500 T cells per mL were only theones with the most profoundimmunologic impairment. Thus, TRECtesting is not a screen for DiGeorgesyndrome, trisomy 21, or any othersyndrome per se. Two-thirds ofpatients with syndromic TCL hadDiGeorge syndrome, usually becauseof interstitial deletion of chromosome22q11.2, but also associated withintragenic heterozygous mutations ofT-box-containing transcription factor1 (TBX1), a transcription factorlocated within the common 22qdeletion region. Additionally, somepatients with DiGeorge syndromelacked a known genetic defect. Incomplete DiGeorge syndrome, somecases of CHARGE syndrome, andsome infants of mothers withdiabetes, profound TCL is due to theabsence of a functional thymus.Urgent intervention to protect suchinfants from infections is requiredas in typical SCID, but curativetreatment requires a thymustransplant, available only byexperimental clinical trial.26,27 All ofthe 4 California infants found by NBSto have complete DiGeorge syndromewho were treated with a thymus

transplant developed functionalT cells, although 1 subsequently diedof an unrelated complication. Infantswith partial DiGeorge syndrome hadstable or rising T cells over time, andthose for whom data were availablemade protective responses to killedvaccines (data not shown).

Thirty-three infants with TCL frompreterm birth alone (Table 7) had.2% of their CD4 helper T cells withthe naïve CD45RA marker, and theirTCL resolved to normal over time.However, longitudinal follow-up wasessential to differentiate them frominfants with SCID who were bornprematurely.

Almost all infants with secondary TCLwere cared for in NICUs because ofrecognized primary conditions,important exceptions being thosewhose mothers had takenimmunosuppressive medication duringpregnancy. Although self-limiting, theeffects of these medications on infantT cells were profound. For example,despite being contraindicated inpregnancy, fingolimod was takenfor multiple sclerosis by 1 womanwho was pregnant. This drug isa sphingosine-1-phosphate receptormodulator that acts to sequesterlymphocytes in the thymus and lymphnodes, preventing their release.37 Theinfant appeared healthy but at 8 daysof age, had only 126 T cells, withundetectable naïve T cells, a profiletypical of SCID that would require HCT.However, further workup revealednormal T-cell proliferation, and by theage of 2 months, the immune profilehad completely resolved withouttreatment.

Out of an initial total of 55 idiopathicTCL cases, 22 defined syndromeswere eventually diagnosed, mostcommonly DiGeorge syndrome in9 case patients who did notdemonstrate the commonly observedneonatal cardiac disease,hypocalcemia, or dysmorphicfeatures. AT was also found in 5 casesthat, without NBS, would have most

likely been diagnosed only afterthe appearance of neurologicmanifestations, ocular telangiectasias,or frequent infections.28 Theremaining syndromes initiallyidentified as idiopathic TCL by NBSincluded 3 cases of diabeticembryopathy and 1 each of CHARGEsyndrome; congenital, lipomatous,overgrowth, vascular malformations,epidermal nevi, and spinal and/orskeletal anomalies and/or scoliosis(CLOVES) syndrome; Nijmegenbreakage syndrome38; EXTL3deficiency29; and member 2 of Racsubfamily of r GTPases (RAC2)deficiency39 (A.P. Hsu, J.A. Church,et al, unpublished observations). Thelast 3 were diagnosed after whole-exome sequencing and analysis.

CONCLUSIONS

As an early adopter of TRECscreening, the California NBS programhas demonstrated not only detectionof SCID and clinically important non-SCID TCL with near-completesensitivity and outstanding specificitybut also implementation of high-throughput, DNA-based methodologyapplicable to other conditions, suchas spinal muscular atrophy. Thepotential harm of live attenuatedrotavirus vaccination in infants whoare immunodeficient has beenaverted, strengthening this otherwisebeneficial public health measure. Theincidence of SCID has proven higherthan originally thought, and newdisease genes have been discoveredin infants identified by SCID NBS.Treatment of SCID detected by NBShas led to 94% survival whilerevealing areas for future furtherimprovement in targetedchemotherapy and avoidance ofCMV infection.

ACKNOWLEDGMENTS

We thank Fred Lorey for his energyand vision in bringing about SCIDscreening in California. Importantsupport and encouragement for us

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and SCID families came from Fredand Vicki Modell, the SCID Angels forLife, and the Immune DeficiencyFoundation.

ABBREVIATIONS

ADA: adenosine deaminaseAT: ataxia telangiectasiaBCL11B: B cell lymphoma 11BCDPH: California Department of

Public HealthCHARGE: coloboma of the eye,

heart defect, atresiachoanae, restrictedgrowth anddevelopment, genitalabnormality, and earabnormality

CI: confidence interval

CLOVES: congenital lipomatousovergrowth, vascularmalformations,epidermal nevi, andspinal and/or skeletalanomalies and/orscoliosis

CMV: cytomegalovirusDBS: dried blood spotERT: enzyme replacement therapyEXTL3: exostosinlike

glycosyltransferase 3GA: gestational ageGDL: Genetic Disease LaboratoryGT: gene therapyHCT: hematopoietic cell transplantIgG: immunoglobulin GIL2RG: interleukin-2 receptor

g chainIL7R: interleukin-7 receptor

JAK3: Janus kinase 3NBS: newborn screeningNK: natural killerPIDTC: Primary Immune

Deficiency TreatmentConsortium

RAC2: member 2 of Rac subfamilyof r GTPases

RAG1: recombinase-activatinggene 1

RAG2: recombinase-activatinggene 2

RMRP: RNA component ofmitochondrial RNAprocessingendoribonuclease

SCID: severe combinedimmunodeficiency

TCL: T-cell lymphopeniaTREC: T-cell receptor excision

circle

and led the California severe combined immunodeficiency newborn screening program, for which Dr Koupaei was the Genetic Disease Laboratory Director in

charge of T-cell receptor excision circle screening; Ms Grimbacher and Dr Nguyen collected and analyzed data and reviewed the manuscript; Dr Naides provided the

analysis of flow-cytometric data and reviewed and revised the manuscript; and all authors approved of the final manuscript as submitted and agree to be

accountable for all aspects of it.

DOI: https://doi.org/10.1542/peds.2018-2300

Accepted for publication Nov 7, 2018

Address correspondence to Jennifer M. Puck, MD, Division of Allergy, Immunology, and Blood and Marrow Transplantation, Department of Pediatrics, University of

California, San Francisco, Box 3118, 555 Mission Bay Blvd S., Room SC-252K, San Francisco, CA 94143. E-mail: jennifer.puck@ucsf.edu

PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275).

Copyright © 2019 by the American Academy of Pediatrics

FINANCIAL DISCLOSURE: The authors have indicated they have no financial relationships relevant to this article to disclose.

FUNDING: Dr Puck received support from grant R01 AI105776 and cooperative agreement U54 AI082973 for the Primary Immune Deficiency Treatment Consortium,

a member of the Rare Diseases Clinical Research Network funded by the National Institute of Allergy and Infectious Diseases, the Office of Rare Diseases Research,

the National Center for Advance Translational Sciences, and the National Institutes of Health; the Jeffrey Modell Foundation; the Lisa and Douglas Goldman Fund; and

the Michelle Platt-Ross Foundation. Drs Butte and Church received support from the Jeffrey Modell Foundation. Dr Dvorak received support from grant U54

AI082973. Funded by the National Institutes of Health (NIH).

POTENTIAL CONFLICT OF INTEREST: Dr Markert developed technology for RVT-802, which has been licensed to Enzyvant Therapeutics GmbH. Dr Markert has received

royalties from Enzyvant. Portions of Dr Markert and her research team’s salaries are being paid by the funding from Enzyvant. If the technology is commercially

successful in the future, Dr Markert and Duke University may benefit financially. Dr Naides is employed by Quest Diagnostics. Donald Kohn is an inventor of

a lentiviral vector for gene therapy of adenosine deaminase severe combined immunodeficiency, which Orchard Therapeutics Ltd has licensed from the University

of California Regents; Dr Kohn is a consultant to Orchard Therapeutics Ltd and a member of its Scientific Advisory Board. Dr Cowan serves on the Scientific Advisory

Boards for Homology Medicine, Inc, and Exogen, Inc, and on the Data and Safety Monitoring Board for bluebird bio, Inc. Dr Puck discloses spousal employment at

a clinical DNA sequencing company, Invitae; the other authors have indicated they have no potential conflicts of interest to disclose.

PEDIATRICS Volume 143, number 2, February 2019 61 by guest on July 1, 2020www.aappublications.org/newsDownloaded from

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