A Simple, High-Throughput Assay for Fragile X Expanded Alleles Using Triple Repeat Primed PCR and Capillary Electrophoresis Elaine Lyon, Thomas Laver,

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A Simple, High-Throughput Assay for Fragile X Expanded Alleles Using Triple Repeat Primed PCR and Capillary Electrophoresis Elaine Lyon, Thomas Laver, Ping Yu, Mohamed Jama, Keith Young, Michael Zoccoli, Natalia Marlowe The Journal of Molecular Diagnostics Volume 12, Issue 4, Pages 505-511 (July 2010) DOI: 10.2353/jmoldx.2010.090229 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Representative screening electropherograms for previously genotyped Coriell samples with normal or expanded CGG alleles. Genotypes are noted below. The threshold cutoff for normal alleles of 55 repeats is shown. A sample with a ladder motif extending beyond the 55 repeat threshold was considered to have an expanded allele. CGG repeat sizes A: Coriell NA06891, premutation male with 118 CGG repeats. B: Coriell NA11880, normal female heterozygote with 29 and 30 repeats. C: Coriell NA12548, normal female homozygote with 30 repeats. D: Coriell NA06910, premutation female heterozygote with 30 and 88 repeats. E: Coriell NA20239, premutation female heterozygote with 20 and 193 repeats. F: Coriell NA04025, full-mutation male with ∼645 repeats. G: No template. The Journal of Molecular Diagnostics 2010 12, 505-511DOI: (10.2353/jmoldx.2010.090229) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Screening electropherograms for intermediate allele (45–54) samples. Samples were previously genotyped by sizing and/or Southern blot. A: 30 and 50 repeats. B: 31 and 45 repeats. C: 47 repeats. D: 29 and 54 repeats (inset: rescaled y axis). The Journal of Molecular Diagnostics 2010 12, 505-511DOI: (10.2353/jmoldx.2010.090229) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Screening electropherograms for mock mosaic dilutions. Coriell NA04025 (full-mutation male) was serially diluted twofold with Coriell NA12548 (normal female) and then processed with the FX Screening assay. The electropherograms were rescaled to visualize the difference between a “stutter” and baseline. A: Undiluted NA12548 (normal) 30 ng/rxn. B: Undiluted NA04025 (full mutation) 30 ng/rxn. C: First dilution—each Coriell at 15 ng/rxn. D: Second dilution—Coriell NA04025 at 7.5 ng/rxn and NA12548 at 22.5 ng/rxn. E: Third dilution—Coriell NA04025 at 3.75 ng/rxn and NA12548 at 26.25 ng/rxn. The Journal of Molecular Diagnostics 2010 12, 505-511DOI: (10.2353/jmoldx.2010.090229) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Feasibility of using dried blood spots as a sample source the FX Screening assay. One 6-mm punch from each blood spot was extracted on the Qiagen BioSprint96 following the standard protocol. A: Female intermediate. B: Female premutation. C: Male full mutation. D: Male intermediate. E: Male normal. F: Female full mutation. The Journal of Molecular Diagnostics 2010 12, 505-511DOI: (10.2353/jmoldx.2010.090229) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 Proposed testing strategy for the implementation of the TRP PCR assay for population screening. The model describes the algorithm which uses the TRP PCR assay as a first-tier test to decrease the number of samples that require diagnostic testing (sizing by PCR or Southern blot) from 50,000 to 660 samples based on the frequency of premutation and full-mutation carriers in the general population. The Journal of Molecular Diagnostics 2010 12, 505-511DOI: (10.2353/jmoldx.2010.090229) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions