A Novel Assay for Evaluating Fragile X Locus Repeats

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Presentation transcript:

A Novel Assay for Evaluating Fragile X Locus Repeats Karl Adler, J. Kent Moore, Galina Filippov, Shaoping Wu, Jon Carmichael, Mack Schermer The Journal of Molecular Diagnostics Volume 13, Issue 6, Pages 614-620 (November 2011) DOI: 10.1016/j.jmoldx.2011.06.002 Copyright © 2011 Terms and Conditions

Figure 1 Results of the hybridization assay and demonstration of microfluidic electrophoresis confirmation with panels of reference samples. A: Hybridization assay response curve for the eight female reference samples. B: The response curve for the eight male reference samples. C–H: Microfluidic electrophoresis results for three of the female references (normal, near the premutation cutoff, and a large full mutation) (C, NA 07540; E, CRL-2704D; and G, NA 05847) and three male references of similar repeat lengths (D, NA 07539; F, NA 20230; and H, NA 04025). The Journal of Molecular Diagnostics 2011 13, 614-620DOI: (10.1016/j.jmoldx.2011.06.002) Copyright © 2011 Terms and Conditions

Figure 2 Histogram of the 997 hybridization assay results from female DBS samples. The individual bin values are scaled on the left axis, and the cumulative percentage line is scaled on the right axis. Table 1 provides the corresponding summary results in detail. The Journal of Molecular Diagnostics 2011 13, 614-620DOI: (10.1016/j.jmoldx.2011.06.002) Copyright © 2011 Terms and Conditions

Figure 3 Detailed electropherograms from two reflex tests of DBS samples using the microfluidic electrophoresis instrument. The assay kit standards at 50 and 10,380 bp bracket the sample peaks in the middle. The electropherogram from the ATCC 31 to 59 reference sample result is overlaid (blue) on both of the unknown samples (red). A: The long allele of sample 126 confirmed to 53 repeats, just less than the premutation cutoff and visibly <59 repeats of the standard. B: The long allele of sample 526 confirmed to 75 repeats, easily visible as>59 repeats of the standard. FU, follow-up. The Journal of Molecular Diagnostics 2011 13, 614-620DOI: (10.1016/j.jmoldx.2011.06.002) Copyright © 2011 Terms and Conditions

Figure 4 A graphical comparison of selected hybridization assay results with the corresponding microfluidic electrophoresis reflex testing results. This includes all of the samples that were tested by electrophoresis: the samples called as ≥45 by the hybridization assay plus 29 randomly chosen samples that were called normal. The dotted line is the theoretical locus where the results of the hybridization assay and the electrophoresis would be identical; it is not a regression line fit to the data. Data points from the eight confirmed premutations (red) and the confirmed intermediates between 45 and 54 repeats (yellow) are shown. Normal data points are shown in blue. By the preponderance of points laying below the dotted line, the hybridization assay is shown to overestimate the number of repeats on average in the intermediate and premutation range. The Journal of Molecular Diagnostics 2011 13, 614-620DOI: (10.1016/j.jmoldx.2011.06.002) Copyright © 2011 Terms and Conditions