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 1
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 2
Information Resources_NCBI 3
NCBI_OMIM Information includes: Clinical Features Diagnosis Clinical Management Pathogenesis Molecular Genetics Genotype/Phenotype Correlations Population Genetics 4
NCBI_PubMed 5
NCBI_Gene 6
NCBI_Gene 7
Information Resources_GeneTests Information includes: Summary Diagnosis Clinical Description Differential Diagnosis Management Genetic Counseling Molecular Genetics Resources 8
HGMD_GALT 9
GALT Mutation Map 10
General Guidelines for PCR_1 Preventing contamination Cross-contamination Carryover contamination Important reaction components DNA polymerase Templates Primers Deoxynucleoside triphosphate (dntps) Magnesium ions 11
General Guidelines for PCR_2 Cycling parameters Denaturing Annealing Elongation PCR products detection Agarose gel electrophoresis 12
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 13
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 14
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. 15
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 16
Tetra-primer ARMS-PCR Reaction Reaction Mix: (25 µl) Thermal Cycler Condition 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 MgCl 2 2.5 mm Taq DNA polymerase 2.5 U Genomic DNA 4 µl 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 17
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. 18
GALT mutations Greg Kopish 19
MSUD Y438N Mutations Greg Kopish 20
TRECs are reduced in nearly all forms of SCID 21
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 22
TaqMan Probe Real-time qpcr REAL-TIME Quantitative PCR (RT-qPCR) 23
TaqMan Probe Real-time qpcr 24
TREC measurement using RT-qPCR 25
Multiplexing the 384-well Plate
Scientific Methodology NBS Card (NSC) a.k.a. Guthrie Card Dried blood spots (DBS) Extract DNA Amplify TREC by real-time QPCR Analyze 3 mm punch 96 well plate Amplification Plot TREC plasmid Standards Rn (amplification ) ABI 7900HT Fast Real-Time PCR System 27
Thank you! Questions? Mei Baker mwbaker@wisc.edu 28