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SCID SCREENING: A NEW YORK STATE OF MIND
New York Newborn Screening Program - DNA
Jason Isabelle June 4-5, 2012
What is SCID? Severe Combined Immunodeficiency
Caused by diverse mutations in several different genes resulting in a combined immune deficiency X-Linked/Autosomal Recessive Inheritance
Prevalence: ~1:40,000-100,000 3-7 affected newborns in NY each year
Extreme lack of T lymphocyte differentiation and function Severally impaired humoral/cellular immunity
Often fatal within the first year of lifePrepared by Jason Isabelle-NYSNSP
SCID Classification
X-linked SCID Mutations in the gene encoding the common gamma chain
of IL-2,4,7, & 9 cytokine+ receptors
Autosomal Recessive SCID Adenosine deaminase deficiency Jak3 tyrosine kinase deficiency RAG1,2 IL-7R (α chain) CD-45
David Vetter: X-linked SCID
Treatment Methods
Allogeneic hematopoietic stem cell transplant (HSCT) Donor marrow is depleted of T cells (Prevents GVHD) Allows for half-matched donor Climbing to a 90% success rate if administered <3 months of age
Enzyme replacement therapy ADA deficient SCID
Gene Therapy X-linked or ADA deficient SCID
T Cell Receptor Excision Circles
By-products of T cell receptor gene rearrangements during T cell maturation in the thymus
TRECs do not replicate during mitosis Episomal DNA that gets diluted by cell divisions
TREC levels in peripheral blood reflect T cell production in the thymus
Low/No TRECs = Low/No T cell production by the thymus
δRec-ψJα TREC Is Ideal
Created from the sequential rearrangements of the TCR α/δ locus 70% of thymocytes that express α/β TCR will form this specific TREC
Signal joint region of this TREC is flanked by a conserved region Allows for universal primer design that will always detect this TREC
Occurs late in maturation Likely to generate a functional and diverse T cell repertoire
Sequential Rearrangements of the TCRAD Locus
Prepared by Jason Isabelle-NYSNSP
Hazenberg MD, Verschuren MCM,Hamann D, Miedema F, van Dongen JMJ (2001) T cell receptor excision circles as markers for recent thymic emigrants: basic aspects, technical approach, and guidelines for interpretation. J Mol Med 79:631-640
History of SCID Testing Automated assay developed and validated 12/2009-9/2010
Submitted validation package to NYS Clinical Laboratory Evaluation Program (CLEP) for approval on 9/08/2010
CLEP and emergency regulation approved 9/27/2010
SCID screening started 9/29/2010
1st “True SCID” baby detected 12/27/2010 (NICHD Support)
Presumptive Positive (Borderline) category added 1/25/2011
Commissioner of Health officially adds SCID to NSP panel 4/12/2011
DNA Extraction Fast Facts
CASM ‘Homebrew’ Extraction
4 Solutions
100μl Total Volume
Tip Intensive
Easily Scalable Low-Mid Throughput
Average Yield: 4-5ng/μl
DNA Extraction Components
RBC Lysis Solution Targets and destroys known PCR inhibitors
▪ Immunoglobulins, hemoglobin, etc
Wash Solution Eliminate lysis by-products
Buffer A Lyses of WBC’s and “scratches” fiber matrix
Buffer B Neutralize pH and solubilize DNA
Why Automate?
Accommodate increased throughput
Reduce repetitive stress injuries
Address staffing shortages
Increase reproducibility and consistency of results
Automation Obstacles
Many manual processes are difficult to automate Centrifugation, heating/cooling, vortexing, etc…
Spot/Tip interactions 10….960….3,840….11,520
Automated Method Development
Labware Adjustments
Liquid Type Editor
Pipetting Template
Volume Conditioning
METHOD CREATION
Liquid Handling System
Each complete Liquid Handling System is capable of 1200 DNA extractions per 8 hour day.
Liquid Handling Components
Each Cytomat has a capacity of 6048 Tips when fully loaded. Each Shaking Peltier Device can heat/cool from +4°C to +70°C .
SCID Assay
10 μl Final Volume (8μl Reaction Mix/2μl DNA)
RNaseP/TREC Multiplex
8-Point Standard Curve In Triplicate 2000,1000,500,250,125,62.5,31.2,15.6 Copies
+SCID/-SCID Control In Triplicate ADA, IL7R alpha, X-Linked, Omenn’s Syndrome—0 Avg
Cutoff: 200 Trecs/μl of Whole Blood
Taqman Chemistry
Reporter/Quencher
5’ Nuclease Activity
Probe Cleavage
Sequence Specific
Multiplex Capability
Applied Biosystems
TREC Assay PCR Product
62bp amplicon
Probe sequence spans signal joint
Beckman Biomek NX
96 Well Extraction Plate to 384 Well Reaction Plate. Shaking-Heating-Multiplate Adapters
Applied Biosystem 7900HT
Each 7900HT is capable of running 1500 samples per 8 hour day.
Absolute Quantification Run
Data Analysis
QPCR TERMS
Baseline Adjustment
Threshold Adjustment
Algorithm Settings
Individual Trace Analysis
Sample PassesApplied Biosystems
“Typical” Amplification Curve
Amplification Curve: Fail!
ADA Deficient SCID
2nd most common form of SCID ~15% of all SCID cases
Autosomal recessive inheritance
Mutations in the ADA gene reduce or eliminate the activity of the enzyme adenosine deaminase Toxic buildup of deoxyadenosine ensues
Massive reduction in lymphocyte population
Affected newborns have options
ADA-SCID Absolute Quantification Run
ADA
RNaseP
TREC
Duplex Amplification Plots
TREC AMPLIFICATION PLOT RNASE P AMPLIFICATION PLOT
ADA
ADA
NY-NBS SCID AlgorithmDried Blood Spot Specimen
Multiplex PCR (TREC/RNaseP)
TREC ≥ 200and
RNase P < WAL
SCREEN NEGATIVE
RNase P ≥ 35
TREC< 200
Sample is retested in duplicate
Two of Three RNaseP WALANDTwo of Three TREC ≥ 200ORAverage of Three TREC ≥200
SCREEN NEGATIVE
Two of Three RNaseP WALANDTwo of Three TREC < 200ANDAverage of Three TREC >125<200AND Gestation Age ≥37AND Has never been a PP before
PRESUMPTIVE POSITIVE
Two of Three RNaseP WALANDTwo of Three TREC <200ANDAverage of Three TREC <200ANDGestation Age <37
REPEAT PREMATURE
Two of Three RNaseP WALANDTwo of Three TREC ≤200ANDGestation Age ≥37ANDAverage of Three TREC ≤200 if a previous PPORAverage of Three TREC <150 if an initial Specimen
REFERRAL
TREC values are copies/ul RNaseP values are Cq
Conclusion
Early detection benefits
Adaptable screening methodology
Prevalence
Treatment
Testing
Funding Support
The New York State Department of Health
The Eunice Kennedy Shriver Institute for Child Health and Human Development
Jeffrey Modell Foundation