A Pseudo-Full Mutation Identified in Fragile X Assay Reveals a Novel Base Change Abolishing an EcoRI Restriction Site Shujian Liang, Harold N. Bass,

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A Pseudo-Full Mutation Identified in Fragile X Assay Reveals a Novel Base Change Abolishing an EcoRI Restriction Site Shujian Liang, Harold N. Bass, Hanlin Gao, Caroline Astbury, Mehdi R. Jamehdor, Yong Qu The Journal of Molecular Diagnostics Volume 10, Issue 5, Pages 469-474 (September 2008) DOI: 10.2353/jmoldx.2008.080059 Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Restriction map surrounding the FMR1 gene illustrating the predicted fragments on Southern blots. Numbering refers to GenBank L29074.1. a: Probe used in all Southern blots (Chemicon International). b: Fragment size predicted with PstI digestion only. c: Fragment size predicted with EcoRI and NruI (a methylation-sensitive enzyme, cutting only unmethylated active X chromosome) double digestion, generating 2.8-kb (on active X chromosome) and 5.2-kb (on inactive X chromosome) fragments. d: EcoRI digestion only, generating either a 5.2-kb fragment (in a normal individual) or a 10.9-kb fragment if the 11,114 EcoRI cutting site is abolished. R, EcoRI restriction site; P, PstI restriction site; SF (special forward primer) and SR (special reverse primer), location of PCR primers designed to bracket the 11,114 EcoRI restriction site (from nucleotide position 10,993 to 11,292). The Journal of Molecular Diagnostics 2008 10, 469-474DOI: (10.2353/jmoldx.2008.080059) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Standard Southern blot of the family. Lanes 2, 5, and 9 are from the index patient (IP), mother (M), and father (F), respectively. Lanes 10 and 11 are the normal (NC) and abnormal controls (AC; Coriell GM07537A). Other lanes represent results from the same batch run from other patients. MW, the Lambda/HindIII MW marker (Chemicon International). The Journal of Molecular Diagnostics 2008 10, 469-474DOI: (10.2353/jmoldx.2008.080059) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Determination of the origin of the 10.9-kb fragment from the index patient. A: Southern blot using PstI digestion only showing bands of 0.99, 1.03, and 1.08 kb (the largest band approximately 1.1 kb in size). An abnormal control (AC, lane 6) shows a full mutation (Coriell GM07537A), with an additional 2.1-kb band (336 CGG repeats). No CGG expansion was noted in the family study (lanes 2 to 4) or in a normal control (NC, lane 5). MW, the Lambda/HindIII MW marker (Chemicon International). B: Southern analysis of the family digested with EcoRI, with (+) and without (−) NruI. MW, molecular marker as in A; NFC, normal female control; M, mother; IP, index patient; F, father; NMC, normal male control; AC, abnormal control as in A. Note that father and the index patient both showed the 10.9-kb fragment (lanes 6, 7, and 9). C: PCR products with (+) and without (−) EcoRI digestion. MR, 100-bp Molecular Ruler (Bio-Rad); NC, normal control; M, mother; IP, index patient; F, father. The Journal of Molecular Diagnostics 2008 10, 469-474DOI: (10.2353/jmoldx.2008.080059) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Confirmation of a base change at EcoRI cutting site by sequencing. a: Arrow points to the position 11,114, where a G→A transition occurred in the father. b: The index patient is heterozygous, with both G and A. c: Mother has a normal G at the same position. The Journal of Molecular Diagnostics 2008 10, 469-474DOI: (10.2353/jmoldx.2008.080059) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 DNA sequence alignment around the 11,114 EcoRI restriction site with 18 species showing conservation status of the base G (derived from University of California Santa Cruz Genome Bioinformatics, http://genome.ucsc.edu/cgi-bin/hgTracks, last accessed April 10, 2008). The Journal of Molecular Diagnostics 2008 10, 469-474DOI: (10.2353/jmoldx.2008.080059) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions