SCID SCREENING: A NEW YORK STATE OF MIND

1. New York Newborn Screening Program - DNA SCID SCREENING: A NEW YORK STATE OF MIND Jason Isabelle June 4-5, 2012

2. 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 life Prepared by Jason Isabelle-NYSNSP

3. 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

4. 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

5. 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

6. 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

7. Sequential Rearrangements of the TCRAD Locus 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 Prepared by Jason Isabelle-NYSNSP

8. 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

9. DNA Extraction Fast Facts • CASM ‘Homebrew’ Extraction • 4 Solutions • 100μl Total Volume • Tip Intensive • Easily Scalable • Low-Mid Throughput • Average Yield: 4-5ng/μl

10. 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

11. Why Automate? • Accommodate increased throughput • Reduce repetitive stress injuries • Address staffing shortages • Increase reproducibility and consistency of results

12. Automation Obstacles • Many manual processes are difficult to automate • Centrifugation, heating/cooling, vortexing, etc… • Spot/Tip interactions • 10….960….3,840….11,520

13. Automated Method Development • Labware Adjustments • Liquid Type Editor • Pipetting Template • Volume Conditioning METHOD CREATION

14. Liquid Handling System Each complete Liquid Handling System is capable of 1200 DNA extractions per 8 hour day.

15. Liquid Handling Components Each Shaking Peltier Device can heat/cool from +4°C to +70°C . Each Cytomat has a capacity of 6048 Tips when fully loaded.

16. 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

17. Taqman Chemistry • Reporter/Quencher • 5’ Nuclease Activity • Probe Cleavage • Sequence Specific • Multiplex Capability Applied Biosystems

18. TREC Assay PCR Product 62bp amplicon Probe sequence spans signal joint

19. Beckman Biomek NX 96 Well Extraction Plate to 384 Well Reaction Plate. Shaking-Heating-Multiplate Adapters

20. Applied Biosystem 7900HT Each 7900HT is capable of running 1500 samples per 8 hour day.

21. Absolute Quantification Run

22. Data Analysis QPCR Terms • Baseline Adjustment • Threshold Adjustment • Algorithm Settings • Individual Trace Analysis Sample Passes Applied Biosystems

23. “Typical” Amplification Curve

24. Amplification Curve: Fail!

25. 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

26. ADA-SCID Absolute Quantification Run ADA RNaseP TREC

27. Duplex Amplification Plots TREC Amplification plot Rnase p amplification plot ADA ADA

28. NY-NBS SCID Algorithm Multiplex PCR (TREC/RNaseP) TREC values are copies/ul RNaseP values are Cq Dried Blood Spot Specimen TREC ≥ 200 and RNase P < WAL TREC< 200 Sample is retested in duplicate RNase P ≥ 35 SCREEN NEGATIVE Two of Three RNaseP WAL AND Two of Three TREC ≤200 AND Gestation Age ≥37 AND Average of Three TREC ≤200 if a previous PP OR Average of Three TREC <150 if an initial Specimen Two of Three RNaseP WAL AND Two of Three TREC ≥ 200 OR Average of Three TREC ≥200 Two of Three RNaseP WAL AND Two of Three TREC < 200 AND Average of Three TREC >125<200 AND Gestation Age ≥37 AND Has never been a PP before Two of Three RNaseP WAL AND Two of Three TREC <200 AND Average of Three TREC <200 AND Gestation Age <37 SCREEN NEGATIVE PRESUMPTIVE POSITIVE REPEAT PREMATURE REFERRAL

29. Conclusion • Early detection benefits • Adaptable screening methodology • Prevalence • Treatment • Testing

30. Funding Support The New York State Department of Health The Eunice Kennedy Shriver Institute for Child Health and Human Development Jeffrey Modell Foundation