Performance Evaluation of Two Methods Using Commercially Available Reagents for PCR-Based Detection of FMR1 Mutation Jane S. Juusola, Paula Anderson,

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Performance Evaluation of Two Methods Using Commercially Available Reagents for PCR-Based Detection of FMR1 Mutation Jane S. Juusola, Paula Anderson, Fernanda Sabato, David S. Wilkinson, Arti Pandya, Andrea Ferreira-Gonzalez The Journal of Molecular Diagnostics Volume 14, Issue 5, Pages 476-486 (September 2012) DOI: 10.1016/j.jmoldx.2012.03.005 Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Interpretation and comparison of results using different reagents. A full mutation female sample (NA05847) was tested with the Asuragen AmplideX FMR1 reagents (A) and the two Abbott Molecular FMR1 Primer Sets (B–D). A: With the Asuragen reagents, the full mutation female shows a peak in the normal repeat range (NOR; 290 bp; 21 repeats; asterisk) and a peak in the full mutation range (FM/FULL; 1129 bp; >200 repeats; dashed arrow). There are also triplet-repeat specific products that are visible as a ladder motif (solid arrow) in the zoomed-in electropherogram (inset). B: With the Abbott Molecular FMR1 Primer 2 reaction (screening), the full mutation female sample shows a normal allele peak (asterisk) and the triplet-repeat specific products (solid arrow), which are indicative of an expanded allele. C: The Abbott Molecular FMR1 Primer 1 reaction shows a sex-specific peak for the X chromosome (203 bp), with an absence of the Y chromosome peak (170 bp), and a peak in the normal repeat range (255 bp; 21 repeats, asterisk). D: This panel shows that no large fragments (in the 500- to 1500-bp range) were visible on the electropherogram, but a smear that is >1000 bases is visible on the agarose gel (dashed arrow). INT, intermediate. The Journal of Molecular Diagnostics 2012 14, 476-486DOI: (10.1016/j.jmoldx.2012.03.005) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Detection of mosaicism. DNA from a normal female sample was mixed with DNA from a premutation (Pre) or full mutation (Full) female sample to give a final concentration of 50% to 0% expanded allele. The Asuragen AmplideX FMR1 reagents were able to detect full mutation mosaicism down to 5% (A) and premutation mosaicism to 1% (B). The Abbott Molecular FMR1 Primer 2 reaction was able to detect both full mutation (C) and premutation mosaicism (D) down to 25%. A triplet-repeat–specific ladder motif is visible into the premutation region (second gray area in A and B, right-most gray area in C and D). The asterisks represent a nonspecific product that is present in some samples. The arrows represent the detected expanded allele. The Journal of Molecular Diagnostics 2012 14, 476-486DOI: (10.1016/j.jmoldx.2012.03.005) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 . Homozygous normal female sample analysis. A homozygous normal female sample (29 repeats) analyzed with the Asuragen AmplideX FMR1 reagents (A) and the two Abbott Molecular FMR1 Primer Sets (B-D). Both methods show a peak in the normal repeat range (NOR; asterisk) and an absence of the ladder motif following that peak (arrow). The Journal of Molecular Diagnostics 2012 14, 476-486DOI: (10.1016/j.jmoldx.2012.03.005) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Interesting case with electropherograms for a female proband with a normal allele (NOR), a full mutation allele (FM), and mosaicism for a premutation (PRE; 35/162/>200 repeats), her mother with a premutation (19/80 repeats), and maternal grandfather with a premutation (84 repeats). A: The Asuragen AmplideX FMR1 reaction amplifies peaks for each allele and the triplet-repeat–specific products indicative of an expanded allele. B: The Abbott Molecular Primer 2 reaction shows the presence of expanded alleles in each family member as a triplet-repeat ladder motif. The Journal of Molecular Diagnostics 2012 14, 476-486DOI: (10.1016/j.jmoldx.2012.03.005) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions