Severe Combined ImmunodeficiencyNewborn Screening in the Navajo
Jennifer [email protected]
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