Transcript Slide 1

T Cell Receptor Excision Circle (TREC)
Assay for Newborn Screening of SCID
Francis K. Lee, M.Sc, Ph.D.
Senior Service Fellow (Research Microbiologist)
Newborn Screening Translation Research Initiative, CDC
Emeritus Professor of Pediatrics, Emory University School of Medicine
Newborn Screening Molecular Workshop
June 28-30, 2011
National Center for Environmental Health · Division of Laboratory
SciencesScreening and Molecular Biology Branch
Newborn
Overview of SCID – the Condition
 Severe Combined Immunodeficiency (SCID) is characterized by the
absence of both humoral and cellular immunity
 At least 15 different genes known to cause SCID when mutated
 All have profound defects in T lymphocyte differentiation and function
 Maternal antibodies wane during first months of life - affected
infants develop infections (common / opportunistic pathogens)
 Recurrent infections, chronic diarrhea, sepsis, FTT
 Death usually before 1 year of age
 Treatment and prevention of infections can
prolong life but are not curative
 Best hope for SCID patients is Hematopoietic
Stem Cell Transplant before the onset of
infections
SCID has been called
“Bubble Boy Disease”
SCID classification
 X-linked SCID: Mutation in the γ chain common to IL-2, IL-4, IL7, IL-9, IL-17 & IL-21 receptors
 Autosomal Recessive SCID:
Adenosine Deaminase deficiency (20q13.11)
Jak3 tyrosine kinase deficiency (19p13.1)
RAG 1 or 2 defect (11p13)
IL-7R deficiency ( chain) (5p13)
Purine Nucleoside Phosphorylase deficiency (14q13)
MHC II deficiency (16p13, 1q21, 13q)
CD3 and CD3 mutations (11q23)
CD45 deficiency
ZAP-70 deficiency- (2q12)
Artemis (10p)
Mutations in IL2R gamma chain
NHIGRI, NIH: Genbank accession number L19546
TRECs: Reduced in All Forms of SCID
Common Feature: ABSENT/NON-FUNCTIONAL T CELLS
IL2R
JAK3
IL7R
CD45
RAG1
RAG2
ARTEMIS
ADA
Reticular Dysgenesis
SCID, multiple bowel atresias
SCID, congenital abnormalities
Severe DiGeorge Syndrome
CD3 Deficiency
CD8 Deficiency
Severe Ataxia Telangiectasia
TTTTTTTTTTTTT+/T+
T+/-
B+
B+
B+
B+
BBBBB+
B+/B+/B+/B+
B+
B+/-
NKNKNK+
NK+
NK+
NK+
NK+
NKNK+
NK+
NK+
NK+
NK+
NK+
NK+
Unknown genetic
Defect ~5-25%
SCID Meets NBS Criteria
 Prevalence of the disease 1:100,000 or greater
SCID: 1:50,000-1:100,000
 Can the disorder be detected by routine physical exam?
SCID: Baby appears normal at birth.
 Does the disease cause serious medical complications?
SCID: 100% fatal within the first year of life
 Is there a cheap, sensitive and specific screening test?
SCID: Real time PCR to enumerate T cell receptor
excision circles
 Is there a confirmatory test?
SCID: Lymphocyte subpopulation analysis
 Does early detection improve outcome?
SCID: Early HSCT decreases mortality from SCID
Optimal Test to Screen for severe
T cell lymphopenia (SCID)
 Must detect low/absent T cells
 Use existing NBS screening cards
 Inexpensive, sensitive and specific
Low rate of false positive tests
Little need for retesting
• Real Time PCR (RT-PCR): enumeration of T cell receptor
excision circles (TREC - surrogate marker for recently
produced T cells) using DNA extracted from newborn blood
spots collected routinely on all newborns
Overview of TREC Assay for
SCID
The T cell Receptor Excision Circle (TREC)
assay differs from other molecular assays used
in NBS:
 Phenotype assay: TREC is a molecular marker for T cell
production in thymus
 Quantitative assay: require higher level of precision
results influence d by
•
DNA extraction efficiency
•
PCR efficiency
Overview of TREC Assay for SCID (cont.)
 T cell receptor excision circles (TREC) are by-products of the
rearrangement of T cell receptor (TCR) genes during thymocyte
maturation in the thymus
 TRECs are episomal DNA and do not replicate during mitosis
 Peripheral blood TREC levels reflect T lymphocyte production
in the thymus
 TREC Assay: Real Time PCR
 Variations in TREC Assay procedures can be based on:
 Primers and Probes
 DNA extraction procedures
TCR–Delta deletion in rearrangement of T cell receptor
Vδ/
gene
ψЈα Jα1 Jα2 Jα3
Vα Vα
δRecVδ1
≈
≈
≈
Chromosomal 14
TCR α/δ chain loci
Alpha chain
V segments
Cα
Jαn
Dδ/ C
Jδ δ
2
Delta chain
V/D/J segments
Alpha chain
J segments
≈
Vαn
1
Alpha chain
constant region
≈
≈
↓
≈
Chromosomal 14
TCR α chain locus
δRec-ΨJα TREC
≈
Signal
joint
Episomal DNA
(δRec-ΨJα TREC)
≈
Chromosomal 14
TCR α/δ chain loci
≈
↓
δRec-ΨJα Coding joint
↓
Vα–Јα–Cα rearrangement
↓
TCR alpha chain transcription, translation , expression
Orientation of δRec and ΨЈα sequences in genomic DNA
δRec
GTGTCCTCACCCGTGAAA
≈
5’
ΨЈα
GTCCACGGATACGTAGTGGCAC
Orientation of δRec and ΨЈα sequences in TREC DNA
≈
Signal
Joint
5’ -------AAAGGTGCCCACTCCTGTGCACGGTGATGCATAGGCACCTG-------3’
Forward Primer Direction →
← Reverse Primer Direction
3’
Technical Approaches to TREC Assays
Classical
Conventional
DBS DNA
Extraction
DBS DNA
Extraction
TREC
sequence
Amplification
Amplicons
Quantification
CDC
PE
DBS
In Situ
Real time
PCR
DBS
In Situ
PCR
Real
time
PCR
Amplicons
Quantification
TREC Measurement: qPCR
Dried
blood
spots
(DBS)
3 mm
punch
Extract Enumerate
DNA* TRECs by
real-time
qPCR
96 well
plate
NBS Card
Cord Bloods TREC Amplification Plots
5
Fluorescence (dRn)
4
3
2
1
0
0
10
20
Cycles
30
40
50
In Situ Real Time
PCR Assay for TREC
Punch one 2.0 mm discs from
DBS specimen into PCR tubes
Wash with 125 µl of DNA
purification solution S1
(shake for 15 minutes at room temp)
Wash with 125 µl of DNA
elution solution S2
(shake for 5 minutes at room temp)
In Situ Real Time PCR Assay for TREC (cont.)
Discard S2 wash buffer
Add 15 μl of qPCR mastermix
(contains complete mix of primers & probe)
Run qPCR in Stratagene MX3000p:
Cord Bloods TREC Amplification Plots
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5
TREC-HeLa DBS calibrators
y = -3.1409x + 36.607
R² = 0.963
E=108%
4
Fluorescence (dRn)
CT#
45 deg for 5 min, 95 deg for 20 min
45 cycles of [ 95 deg x 15 sec + 60 deg x 1 min ]
3
2
1
0.00
1.00
2.00
log10(cell#/μl blood)
3.00
0
0
10
20
Cycles
30
40
50
Quality Assurance
Use of TREC Reference Materials
National Center for Environmental Health
Newborn Screen and Molecular Biology Branch