New York Newborn Screening Program - DNA Jason IsabelleJune 4-5, 2012

 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  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

 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

 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

 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

 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

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:

 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  1 st “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

 CASM ‘ Homebrew’ Extraction  4 Solutions  100μl Total Volume  Tip Intensive  Easily Scalable  Low-Mid Throughput  Average Yield: 4-5ng/μl

 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

 Accommodate increased throughput  Reduce repetitive stress injuries  Address staffing shortages  Increase reproducibility and consistency of results

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

 Labware Adjustments  Liquid Type Editor  Pipetting Template  Volume Conditioning METHOD CREATION

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

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

 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

 Reporter/Quencher  5’ Nuclease Activity  Probe Cleavage  Sequence Specific  Multiplex Capability Applied Biosystems

 62bp amplicon  Probe sequence spans signal joint

96 Well Extraction Plate to 384 Well Reaction Plate.Shaking-Heating-Multiplate Adapters

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

QPCR TERMS  Baseline Adjustment  Threshold Adjustment  Algorithm Settings  Individual Trace Analysis Sample Passes Applied Biosystems

 2 nd 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 RNaseP TREC

TREC AMPLIFICATION PLOTRNASE P AMPLIFICATION PLOT ADA

Dried Blood Spot Specimen Multiplex PCR (TREC/RNaseP) TREC ≥ 200 and RNase P < WAL SCREEN NEGATIVE RNase P ≥ 35 TREC< 200 Sample is retested in duplicate Two of Three RNaseP WAL AND Two of Three TREC ≥ 200 OR Average of Three TREC ≥200 SCREEN NEGATIVE 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 PRESUMPTIVE POSITIVE Two of Three RNaseP WAL AND Two of Three TREC <200 AND Average of Three TREC <200 AND Gestation Age <37 REPEAT PREMATURE 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 REFERRAL TREC values are copies/ul RNaseP values are Cq

 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