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 T Cell Receptor Excision Circle (TREC) Assay for Newborn Screening of SCID National Center for Environmental Health · Division of Laboratory Sciences Newborn Screening and Molecular Biology Branch
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T Cell Receptor Excision Circle (TREC) Assay for Newborn Screening of SCID
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 . - PowerPoint PPT Presentation
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Francis K. Lee, M.Sc, Ph.D.Senior Service Fellow (Research Microbiologist)
Newborn Screening Translation Research Initiative, CDCEmeritus Professor of Pediatrics, Emory University School of Medicine
T Cell Receptor Excision Circle (TREC) Assay for Newborn Screening of SCID
National Center for Environmental Health · Division of Laboratory SciencesNewborn Screening and Molecular Biology Branch
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
Overview of SCID – the Condition
SCID has been called “Bubble Boy Disease”
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 classification X-linked SCID: Mutation in the γ chain common to IL-2, IL-4, IL-
Prevalence of the disease 1:100,000 or greaterSCID: 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
SCID Meets NBS Criteria
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 testsLittle 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
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
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
Overview of TREC Assay for SCID (cont.)
Alpha chain V segments
Delta chain V/D/J segments
Alpha chain J segments
Alpha chain constant region
TCR–Delta deletion in rearrangement of T cell receptor geneVα