Triplet Repeat Primed PCR Simplifies Testing for Huntington Disease

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Presentation transcript:

Triplet Repeat Primed PCR Simplifies Testing for Huntington Disease Mohamed Jama, Alison Millson, Christine E. Miller, Elaine Lyon The Journal of Molecular Diagnostics Volume 15, Issue 2, Pages 255-262 (March 2013) DOI: 10.1016/j.jmoldx.2012.09.005 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic of partial sequence of exon 1 of the HTT gene illustrates how the reverse HD primer can behave as specific2 or chimeric3 during PCR. Also listed is the sequence and location of primers with sites of known polymorphisms. Illustrations not drawn to scale. Data are from ∗Margolis et al (HD-specific primers; set in bold),18 †Yu et al (HD chimeric primers),17 and ‡Gellera et al8 (CAA deletion). §The forward primer stops at TCC to avoid the polymorphic as indicated. The Journal of Molecular Diagnostics 2013 15, 255-262DOI: (10.1016/j.jmoldx.2012.09.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Screen capture of GeneMarker software showing allele binning of all stuttering generated from Coriell sample NA20253. The vertical lines represent an allele within each specific CAG bin for automated allele calls from the fifth CAG repeat to 110th, clearly seen on the insert. The column in the table labeled “size” shows the mean allele sizes from within and between runs. The left and right ranges represent the bin boundaries calculated from two SDs. The boxes at the top of the screen depict the CAG repeats grouped into normal, mutable, reduced penetrance allele (RPA), and affected alleles. The Journal of Molecular Diagnostics 2013 15, 255-262DOI: (10.1016/j.jmoldx.2012.09.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Electropherogram results of samples. A: Electropherogram result of sample NA20245 showing a homozygous allele call of 15 CAG repeats. B: Electropherogram result of sample NA20207 showing a heterozygous allele call of 19/21. Inserted is a close-up view of stuttering between alleles, allowing both the 19 and 21 alleles to be distinguished. The Journal of Molecular Diagnostics 2013 15, 255-262DOI: (10.1016/j.jmoldx.2012.09.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Electropherogram results. A: Electropherogram of HD genotyping of Coriell sample NA20253 showing alleles of 22/101 CAG repeats. Superimposed close-up electropherogram shows the most prominent peak is in the middle. The sample easily genotypes as 101 CAG repeats. B: Electropherogram result of an expanded allele in sample MGL-14. Note the continuous stuttering after the prominent peak at 19 CAG repeats in the close-up view, an indication of the presence of an expanded allele that is identified but not correctly sized by this assay (see superimposed electropherogram). The shown sample will require Southern blot analysis to determine an approximate size of the expanded allele. The Journal of Molecular Diagnostics 2013 15, 255-262DOI: (10.1016/j.jmoldx.2012.09.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions