New York Newborn Screening Program - DNA
Jason Isabelle
June 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: ~1:40,000-100,000
 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

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
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 79:631640
Prepared by Jason Isabelle-NYSNSP

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

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
Applied Biosystems

Reporter/Quencher

5’ Nuclease Activity

Probe Cleavage

Sequence Specific

Multiplex Capability

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

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
RNaseP
TREC
TREC AMPLIFICATION PLOT
RNASE P AMPLIFICATION PLOT
ADA
ADA
Dried Blood Spot Specimen
Multiplex PCR (TREC/RNaseP)
TREC ≥ 200
and
RNase P < WAL
TREC values are copies/ul RNaseP values are Cq
TREC< 200
Sample is retested in duplicate
RNase P ≥ 35
SCREEN NEGATIVE
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

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