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