Expansion of an FMR1 Grey-Zone Allele to a Full Mutation in Two Generations Isabel Fernandez-Carvajal, Blanca Lopez Posadas, Ruiqin Pan, Christopher Raske,

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Expansion of an FMR1 Grey-Zone Allele to a Full Mutation in Two Generations Isabel Fernandez-Carvajal, Blanca Lopez Posadas, Ruiqin Pan, Christopher Raske, Paul J. Hagerman, Flora Tassone The Journal of Molecular Diagnostics Volume 11, Issue 4, Pages 306-310 (July 2009) DOI: 10.2353/jmoldx.2009.080174 Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Pedigree showing the proband (42-6) family members. CGG repeat numbers were as follows: (42-2), 52 CGG repeats (gray zone) (42-3); 34, 56 CGG repeats (premutation); (42-4) 23 CGG repeats (normal); (42-6) ∼538 CGG repeats (full mutation); (42-7) 23, 91 CGG repeats (premutation). The Journal of Molecular Diagnostics 2009 11, 306-310DOI: (10.2353/jmoldx.2009.080174) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 A: Southern blot analysis of genomic DNA isolated from the proband's parents; his mother (42-3) with a premutation allele, his father (42-4) with a normal allele (lanes 1 and 2, respectively). Proband, with a full mutation is shown in lane 3. His sister a premutation carrier (42-7) (lane 4). A normal female control is shown in lane 5. The normal, unmethylated band (2.8 Kb) and the normal methylated band (5.2 Kb) are indicated. A DNA 1kb ladder is shown in lane 6. B: PCR size analysis using the Qiaxcel capillary system (see: Materials and Methods) of PCR products derived from subject 42-2 (52 CGG repeats) and from subject 42-3 (34, 56 CGG repeats). PCR products derived from clones 56a (53 CGG), 56b (58 CGG), 56c (57 CGG), and 56 d (56 CGG) are also shown. PCR reactions used primers 1 and 3.20 C: PCR size analysis for family members 42-2, 42-3, 42-6, and 42-7 using the ABI 3130 DNA Sequencing technology and the Abbott Molecular Fragile X PCR Test (see: Materials and Methods). In each frame, reference peaks (marker X-Rhodamine [ROX] labeled DNA ladder, size range: 50 to 1000 bp) are indicated in outline (also light gray in 42-3 and 42-7). For 42-2 and 42-3, standards were 300, 350, and 400 bp (36 to 69 CGG repeats); for 42-7, standards were 250, 300, 350, 400, 450, and 475 bp (19 to 94 CGG repeats); for 42-6, upper standard was 1000 bp (269 CGG repeats). For 42-6, the full mutation allele was sized by Southern blot. The Journal of Molecular Diagnostics 2009 11, 306-310DOI: (10.2353/jmoldx.2009.080174) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Sequencing analysis of 42-2 and 42-3. The upper electropherogram shows the sequencing analysis of subject 42-2 with an FMR1 allele comprising 52 CGG repeats and 2 AGG interruptions. The bottom electrophoregram shows the sequence of the cloned allele from subject 42-3 and the absence of AGG interruptions. The Journal of Molecular Diagnostics 2009 11, 306-310DOI: (10.2353/jmoldx.2009.080174) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions