Severe Combined ImmunodeficiencyNewborn Screening in the Navajo

Jennifer PuckPuckJ@peds.ucsf.edu

Department of PediatricsUniversity of California, San Francisco

Severe Combined Immunodeficiency--SCID

• Lack of T and B-cell immunity– Absent or very few T

cells– Absent or

nonfunctional B cells– NK cells may be

present or absent• Recurrent and

opportunistic infections from age 2-4 months

• Early mortality unless immune system restored

Justifications for Newborn Screening

Proposed Screening Criteria How SCID Meets Criteria

Disease is serious

Incidence supports screening

Confirmative testing is well-established

Effective treatment exists

Earlier treatment is better

Diagnosis and treatment are available

Screening is cost-effective

Disease is not detected by exam

Galactosemia = 1/60,000Biotinidase Deficiency = 1/80,000

Newborns with SCID appear healthy

Fatal in early life if untreated

Unknown, estimated at 1/40,000

T cell counts, mitogen responses

Bone marrow transplantation

Best survival and outcome when treated before infections occurPediatric immunology BMT centers

An inexpensive, high-throughout screening test could save many lives

X-linkedSCID

IL2RG

γc

ADAJAK3

IL7Rα

Artemis

Unk

now

n

RAG1/2, CD45, TCRδ/ε/ζ, LIG4, CD45, LCK, STAT5b, FOXN1, CHH, SCID & GI atresias,SCID & anomalies,Retic. dysgenesis 20

Genes for

SCIDin

2008

New SCID Defect: Coronin-1A, regulator of actin cytoskeletonUnusual presence of thymusShiow, Paris, Cyster, Sorensen, Puck. Nat Immunol and Clin Immunol, 2008

Copy Number on Ch 16p11.2 Assayed with Affy 6.0 SNP Chip

NK cells

SCID Gene Defects Reveal Non-redundant Steps in T-cell Development

X

XX X

• Lymphocyte antigen receptor rearrangement– RAG1, RAG2, Artemis (DCLRE1C)

• Antigen receptor engagement and signaling– CD3 components, Lck

• Cytokine signaling via cell surface receptors and their intracellular targets– IL2RG (common gamma chain)– IL7R– JAK3– STAT5

• Purine metabolism– Adenosine deaminase, purine nucleoside

phosphorylase• Others?? 10% of SCID patients not accounted for

Human SCID Gene Pathways

• 1968: 1st successful BMT• Best donor: HLA-matched sib

Haploidentical parentCord bloodUnrelated adult donors

• Best time to treat:Before infections

• Best treatment protocol?PIDTC now established18 large & 19 small North

American centersRetrospective and prospective

natural history studies,eventual comparativetreatment trials

40 Years of Hematopoietic CellTransplants for SCID

Years post-transplantation

SCID Patients Treated with BMT Early Have Better Survival

Years Post-Transplantation

Duke BMT older vs. younger than 3.5 months (R. Buckley)

Survival of SCID Diagnosed Early(< 3 mo, + family history) vs. Later

Puck interview study: 39 SCID cases enrolled via SCID family website posting

17 Athebaskan SCID (SCID-A)Patients with BMT at UCSF

ARTEMIS, or DCLRE1C, is a DNA repair protein1/2000 Navajo births homozygous for Y192X mutation

205 IL2RG Mutations in 351 Unrelated Families with XSCID (62% Puck lab)

29

3 3 3 333 3 36 6 6

14

C

11 130 284 469 609 772 869 939 1124

2 3 4 5 6 7 8

cDNA

Exon

7

no translation

polymorphism

splice

63 365

76 57

17 18

IL2RG Domain

nonsense missense

deletion, frame shift deletion, in frame

large deletioncomplex

poly-A addition site

insertion, frame shift insertion, in frame

Mutation Type

WTM B

W

BTM

signal sequence

3, untranslatedbox1-box2 domaintransmembraneWSXWS boxconserved cysteine

33 33 33 3

A

A

WW

CC CC

IL2RGbase, J. Puck

Mutation Saturation--Are we there yet?

T Cell Receptor Excision Circles (TRECs)

3 mm hole punched from blood spot

3 ul blood

Measure TRECs by

PCR

Extract DNA

50 ul blood

Guthrie Card

TREC Dried Blood Spot Assay

1

10

100

1000

10000

Newborn Dried Blood Spots from Different Sources

Cord Blood Spotted in Lab

Routine Care Nursery

Special Care Nursery

AR SCID Newborn Nursery Card

TREC

/106

bet

a-ac

tin

XSCID Newborn Nursery Card

<30

Undetectable

SCID Dried Blood Spotted at Time of Diagnosis

SCID Diagnosis Missed31 week premature twins initially had lung problems

Boy weaning from O2 & growing, but developed E. coli line sepsis, pneumonia

Died at 3.5 months. CMV virus found in blood and lung

Chart review: low lymphocyte numbers: 570 (cord blood at birth), 360, 168, 468

Younger brother diagnosed with SCID at birth, received early BMT from matched sister

• 1 punch (3 ul) used for DNA extraction (Autogen).• 1/10 of product in duplicate Taqman Quantitative PCR with

albumin; 1st run TRECs only.• Samples with no TRECs (1/300): repeat punch and assay

TRECs and b-actin genomic control.• 1,000 California anonymous DBS: 3 with no TRECs and no

actin: indeterminate.• Second spot obtained for new punches: only 1

indeterminate (1/1,000).• When >100 TREC obtained, sample is considered not

consistent with SCID.• When actin is positive, TREC undetectable, sample is

consistent with SCID.• Assay cost is $6 per sample.

Puck Lab TREC Test

Navajo SCID Screening Pilot Study for 2000 babies

Each hospital screensabout 500 babies/year

1000 babies in 1 year

2000 babies in 2 years

About 1%, or 20 in 2 years, called back for more tests

About 1 in 2000, or 1 in 2 years may have SCID

LEVEL I LEVEL IISCID

or related condition

Navajo SCID Screening-Study Sites and People

Tuba City Hospital•Dr. Hu, NAIHS Study Director•Contractor 1

keep database of enrollees, TREC results,Tuba recontacts, upload Chinle data, back up Cont. 2

•Contractor 2 to present study to parents in L & D, enroll, send DBS to UCSF weekly

Chinle Hospital•Dr. Nix, Assoc. NAIHS Study Director for Chinle area•Contractor 3

present study to new parents, enroll, send DBS, funnel data to Tuba City database, track Chinle area recontacts

•Contractor 4 to back up Cont. 3

UCSF•Dr. Puck, PI; Dr. Cowan, Co-Investigator•Diana Gonzalez, Lab tech: Receive DBS and run TRECs; run actin PCR on samples with low TREC; record results; review with Drs. Puck and Cowan weekly; prepare reports. •Dr. Puck and/or Cowan will review all low-TREC samples and report to Dr. Hu (Nix).•Dr. Puck and/or Cowan will receive all follow-up specimens for clinical lab and repeat TRECs, will arrange testing and report immediately to Dr. Hu (Nix).

Navajo SCID Initial Screening and Reporting

Tuba City Hospital•Maintain field records (Tuba & Chinle)•Interview, consent, file consent forms•Collect spots (label: code#, date time)•Collect identifier data, log in notebook and computer•Monday: Send past week samples to UCSF by FEDEX

Chinle Hospital•Interview, consent, file consent forms•Collect spots (label: code#, date time)•Collect identifier data, log in notebook and computer•Monday: Send past week samples to UCSF by FEDEX

UCSF•Tuesday: receive spots from Tuba & Chinle, make punches and start O/N DNA extraction•Wednesday: finish DNA prep & run TREC Q-PCRs•Thursday: see results, re-punch spots with low TRECs and start 2nd DNA preps. If NO low TRECs, complete report for the week.•Friday: finish 2nd DNA prep and run TREC and actin Q-PCRs•Monday: review previous week; email report for week to Tuba City; if any samples are INDETERMINATE or CONSISTENT WITH SCID, page Dr. Hu and if Chinle Dr. Nix to plan f/u.

Navajo SCID Follow-up Samples and Reporting

Tuba City Hospital•Dr Hu contacts local provider, family•Baby returns for eval and blood samples for UCSF clinicalimmunol lab

Chinle Hospital•Dr. Nix contacts provider, family•Baby returns for eval andblood samples for UCSF clinical immunol lab

UCSF•Monday: INDETERMINATE or CONSISTENT WITH SCID, page Dr. Hu and if Chinle Dr. Nix to plan f/u.•Blood samples FEDEXed to UCSF individually; clinical immunology lab for flow cytometry; Puck lab for blood spot, DNA•Fax results back to Reservation; phone to confirm.

Conclusions• SCID is treatable if diagnosed early.• SCID can be amenable to newborn screening

by TREC assay.• Demonstration pilot projects, such as in the

Navajo, are needed to demonstrate clinical utility.

• Identifying infants through screening who have low TRECs will– Improve SCID patient outcomes– Establish SCID incidence– Make possible identification of further immune

defects

Navajo SCID Test Study

Acknowledgements

SupportUSIDNet UCSF CTSA award from NCRR, NIH, UL RR024131-01Jeffrey Modell FoundationNIH RO3NNSGCCDC

SCID Patients and their families

Immunologists and GeneticistsRebecca Buckley, DukeRicardo Sorensen and Ken Paris, LSU

UCSFMort Cowan, Diane WaraJason Cyster, Larry ShiowNavajo Indian SCID ScreeningDiana Hu, MDKristi Nix, MDCalifornia Dept. of Pub HealthFred Lorey, Marty Kharrazi, Lisa FeuchtbaumNNSGCBrad TherrellNIHKee Chan, Joie Davis

Coronin SCID PatientTable 1. Representative immunological studies of blood lymphocytes. Patient age (mo) 15 (after vaccine-

strain varicella) 23 42-49

WBC/ul 3,200 5,310 3.800 Lymphocytes/ul 900 1,430 950 Lymphocyte subsets: number (%) CD3+ T cells 110 (12) 330 (35) CD4+ T helper 50 (6) 170 (18) CD8+ T cytotoxic 30 (3) 100 (10) CD56/16+, CD3- NK cells 340 (28) 360 (38) CD19+ B cells 410 (45) 250 (26) CD3/45RA %Tcells 67 (7) ND1

CD3/45RO %Tcells 33 (4) ND Immunoglobulins (mg/dl) IgM 46 55 69 IgG NE2 889 NE IgA 42 88 48 IgE 15 10 12 Proliferation studies (cpm) Phytohemagglutinin 38,227 ND 10,381 ConcanavalinA 6,270 ND 26,080 Pokeweed mitogen 4,874 ND 10,219 Specific antibody titers Tetanus toxoid 0.02 ND ND Hemophilus influenzae type B 0.7 ND ND Varicella, IgM specific 0.03 ND ND Pneumococcal serotype 43 <0.08 1.06 ND Pneumococcal serotype 6B <0.08 0.28 ND Pneumococcal serotype 9V <0.08 1.44 ND Pneumococcal serotype 14 0.13 3.02 ND Pneumococcal serotype 18C 0.08 0.6 ND Pneumococcal serotype 19F <0.08 0.2 ND Pneumococcal serotype 23F <0.08 0.95 ND 1Not done. 2Not evaluable; patient may have received immunoglobulin G prior to 15 mo and did receive it after 24 mo of age. 3Patient had received 3 doses of 7-valent, protein conjugated pneumococcal vaccine.

Conclusions1. An Affymetrix custom array gives excellent

performance for hemizygous analysis; very good performance for heterozygous analysis

2. About 90% of SCID cases can be diagnosed with the array plus 2 runs of confirmatory dideoxy sequence

3. Cost of array is ~$1200 for IL2RG, IL7R, RAG1, RAG2, Artemis, and JAK3, far lower than individual dideoxy sequences.

ThanksJanet WarringtonRichard ChilesMort CowanTonya LebetDiana Gonzalez

Jeffrey Modell Foundation and UCSF Jeffrey Modell Diagnostic Center for PID

USIDNET