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 Pediatrics University of Wisconsin School of Medicine and Public Health Science Advisor, Newborn Screening Laboratory Wisconsin State Laboratory of Hygiene

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

Information Resources_NCBI

NCBI_OMIM Information includes: Clinical Features Diagnosis Clinical Management Pathogenesis Molecular Genetics Genotype/Phenotype Correlations Population Genetics

NCBI_PubMed

NCBI_Gene

NCBI_Gene

Information Resources_GeneTests Information includes: Summary Diagnosis Clinical Description Differential Diagnosis Management Genetic Counseling Molecular Genetics Resources

HGMD_GALT

GALT Mutation Map

General Guidelines for PCR_1 Preventing contamination Cross-contamination Carryover contamination Important reaction components DNA polymerase Templates Primers Deoxynucleoside triphosphate (dNTPs) Magnesium ions

General Guidelines for PCR_2 Cycling parameters Denaturing Annealing Elongation PCR products detection Agarose gel electrophoresis

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

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

Scheme of tetra-primer ARMS-PCR Ye, S. et al. Nucl. Acids Res. 2001 29:e88; doi:10.1093/nar/29.17.e88 Copyright restrictions may apply.

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

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

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.

GALT mutations Greg Kopish

MSUD Y438N Mutations Greg Kopish

TRECs are reduced in nearly all forms of SCID

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

REAL-TIME Quantitative PCR (RT-qPCR) TaqMan Probe Real-time qPCR REAL-TIME Quantitative PCR (RT-qPCR)

TaqMan Probe Real-time qPCR The geometric increase in fluorescence corresponds to the exponential increase in product and is used to determine the threshold cycle or Ct in each reaction Relative [DNA] during the exponential phase of a reaction is determined by plotting fluorescence against cycle number on a logarithmic scale (so an exponentially increasing quantity will give a straight line).

TREC measurement using RT-qPCR

Multiplexing the 384-well Plate

Dried blood spots (DBS) Amplify TREC by real-time QPCR NBS Card (NSC) a.k.a. Guthrie Card Dried blood spots (DBS) 3 mm punch 96 well plate Extract DNA Amplify TREC by real-time QPCR Analyze Scientific Methodology ∆Rn (amplification) Amplification Plot TREC plasmid Standards ABI 7900HT Fast Real-Time PCR System

Mei Baker mwbaker@wisc.edu Thank you! Questions? Mei Baker mwbaker@wisc.edu