1WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE

Molecular LDT in Newborn Screening Laboratories

APHL/CDC Newborn Screening Molecular Workshop Atlanta, GA

June 28-30, 2011

Mei Baker, M.D., FACMG

Assistant Professor, Department of PediatricsUniversity of Wisconsin School of Medicine and Public Health

Science Advisor, Newborn Screening LaboratoryWisconsin State Laboratory of Hygiene

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Outline

• Background information on the disorder

• Knowledge of the gene and disease-causing mutations

• Assay development and validation

– PCR in general– TETRA-primer ARMS-PCR----GALT mutation analysis– Rea-time qPCR----screening for SCID by quantitating

TRECs

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Information Resources_NCBI

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NCBI_OMIM

Information includes:Clinical Features DiagnosisClinical Management PathogenesisMolecular GeneticsGenotype/Phenotype CorrelationsPopulation Genetics

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NCBI_PubMed

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NCBI_Gene

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NCBI_Gene

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Information Resources_GeneTests

Information includes:SummaryDiagnosisClinical DescriptionDifferential DiagnosisManagementGenetic CounselingMolecular GeneticsResources

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HGMD_GALT

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GALT Mutation Map

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General Guidelines for PCR_1

• Preventing contamination– Cross-contamination– Carryover contamination

• Important reaction components– DNA polymerase– Templates– Primers– Deoxynucleoside triphosphate (dNTPs)– Magnesium ions

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General Guidelines for PCR_2

• Cycling parameters– Denaturing– Annealing– Elongation

• PCR products detection– Agarose gel electrophoresis

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Utilities of Molecular Testing in NBS

• Molecular assay as the second tier testing

biochemical metabolites or enzyme activities can be influenced by feeding history, sampling time and environmental factors.

Timely gene mutation information can be helpful in disease management.

• Molecular assay as the primary screening testing

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Utilities of Molecular Testing in NBS: Examples

• GALT gene mutations are responsible for classic galactosemia, and the common mutations are Q188R, S135L, K285N, L195R, Fl71S and Y209C.

• N314D Duarte variant.

• BCKDHA Y438N is only MSUD mutation in the Old Order Mennonite population, which is increasing in the state of Wisconsin. In this population, classic MSUD has a frequency as high as 1 in 176 births

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15Copyright restrictions may apply.

Ye, S. et al. Nucl. Acids Res. 2001 29:e88; doi:10.1093/nar/29.17.e88

Scheme of tetra-primer ARMS-PCR

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Primer Design http://cedar.genetics.soton.ac.uk/public_html/primer1.htm

Source sequence (up to 1,000 bases)

Position of SNP from start of sequence

Allele 1

Allele 2

Optimum (inner) product size

Maximum (inner) product size

Minimum (inner) product size

Maximum relative size difference of two inner products

Minimum relative size difference of two inner products

Shu Ye, Nucleic Acid Research, 2001, Vol. 29, No. 17, E88-8

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Tetra-primer ARMS-PCR Reaction

• Reaction Mix: (25 µl)

1X PCR buffer Forward inner primer 10 pmol Reverse inner primer 10 pmol Forward outer primer 1 pmol Reverse outer primer 1 pmol DNTPs 200 µM MgCl2 2.5 mM Taq DNA polymerase 2.5 U Genomic DNA 4 µl

• Thermal Cycler Condition

1. 95ºC for 5 minutes 2. 95ºC for 30 second 3. 64ºC for 30 second 4. 72ºC for 40 second 5. repeat 2-4 for 32 cycles 6. 72ºC for 2 minutes

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Procedure

DNA purification from a 1/8” dried blood spot (45 minutes)↓

Tetra-primer ARMS-PCR set-up (30 minutes)↓

Tetra-primer ARMS-PCR amplification (90 minutes)↓

Agarose gel electrophoresis (60 minutes)↓

Gel photography under Blue Light and data analysis (15 minutes)

Notes: 1. The assays for different mutations are performed simultaneously using the same conditions. 2. It costs about $3.00 per genotyping in terms of consumables and reagents.

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GALT mutations

Greg Kopish

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MSUD Y438N Mutations

Greg Kopish

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TRECs are reduced in nearly all forms of SCID

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T Cell Receptor Recombination During Development in the

Thymus

Generation of T cell receptor excision circles (TRECs) occur in >70% of all new (naïve) T cells and can be detected by

PCR

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TaqMan Probe Real-time qPCR

REAL-TIME Quantitative PCR (RT-qPCR)

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TaqMan Probe Real-time qPCR

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TREC measurement using RT-qPCR

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Multiplexing the 384-well Plate

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NBS Card(NSC)

a.k.a. Guthrie Card

Dried blood spots (DBS)

3 mm punch 96 well plate

ExtractDNA

Amplify TREC by real-time

QPCR

Analyze

Scientific Methodology

∆R

n

(am

plifi

cati

on

)

Amplification Plot

TREC plasmid

Standards

ABI 7900HT Fast Real-Time PCR System

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Questions?

Mei Baker

mwbaker@wisc.edu

Thank you!