Syed Hussain Askree, Lawrence N

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Allelic Dropout Can Cause False-Positive Results for Prader-Willi and Angelman Syndrome Testing Syed Hussain Askree, Lawrence N. Hjelm, Muhammad Ali Pervaiz, Margaret Adam, Lora J.H. Bean, Madhuri Hedge, Bradford Coffee The Journal of Molecular Diagnostics Volume 13, Issue 1, Pages 108-112 (January 2011) DOI: 10.1016/j.jmoldx.2010.11.006 Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 A: Schematic map of the PWCR. Physical map of the SNRPN, UBE3A, and snoRNA HBII-85 loci. Also shown is the general layout of the sequence corresponding to the primers and restriction sites for MSP and Southern blot analyses, respectively. B: MSP analysis of SNRPN DNA of patient samples. PCR products of 174 bp and 100 bp are amplified from methylated and unmethylated alleles of the SNRPN DNA locus, respectively. The samples are as follows: lane 1, 100-bp NEB ladder; lanes 2 and 3, patient samples (lane 3 contains the sample with allelic dropout); lane 4, negative control; lane 5, PWS positive control; lane 6, AS positive control; lanes 7 and 9 are empty; lane 8, no template control; and lane 10, untreated genomic DNA control. C: Southern analysis of SNRPN DNA of patient samples. Restriction fragment analysis of patient DNA digested with the methylation-sensitive enzyme KspI and the methylation-insensitive enzyme XbaI. The methylated maternal-specific band is 4.2 kb, and the unmethylated paternal-specific band is 1.1 kb. The samples are as follows: lanes 1 and 8, NEB 1-kb ladder; lanes 2 and 3, patient samples (lane 2 contains the sample with allelic dropout and lane 3 contains a sample from a PWS patient); lane 4, negative control; lane 5, PWS positive control; lane 6, empty; lane 7, AS positive control. The Journal of Molecular Diagnostics 2011 13, 108-112DOI: (10.1016/j.jmoldx.2010.11.006) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Nucleotide change detected in the primer sequence specific for amplifying unmethylated SNRPN DNA in a patient sample. Electropherogram demonstrating a heterozygous G to A change in the SNRPN DNA of the proband (in bold, marked with the double headed arrow), corresponding to the next-to-last base of the reverse primer sequence (PR2) used in amplifying the unmethylated allele in the MSP. The Journal of Molecular Diagnostics 2011 13, 108-112DOI: (10.1016/j.jmoldx.2010.11.006) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 A: SNRPN DNA sequence with primers used for MSP analysis in testing for PWS and AS. The sequence of the SNRPN promoter that displays differential methylation is shown from position 22751100 to 22751646. The top line represents the genomic reference sequence. The second line represents the maternal methylated sequence, and the third line the paternal unmethylated sequence after sodium bisulfite treatment. The CpG dinucleotides are shown in red. Sequences highlighted in gray correspond to primers that were designed originally.11 Sequences highlighted in yellow correspond to the alternative primers designed during this study. Sequences highlighted in magenta correspond to primers that amplify unmodified DNA. The G highlighted in green indicates the location of the SNP causing allelic dropout. B: MSP analysis for PWS and AS using the Kubota et al primer set and an alternative primer set. Top panel: Sodium bisulfite-treated DNA was amplified with original primer sets designed by Kubota et al.11 The bands between the 100-bp band and the 174-bp band are observed frequently with this assay, and we attribute these bands to nonspecific amplification of other regions in the sodium bisulfite-treated genomic DNA. Bottom panel: The same sodium bisulfite-treated DNAs were amplified with the alternative primer set. Lane 1, 100-bp ladder; lanes 2 to 4, patient samples (lane 4 contains the sample with allelic dropout); lane 5, negative control; lane 6, PWS positive control; lane 7, AS positive control; lane 8, no template control; lane 9, untreated genomic DNA. The Journal of Molecular Diagnostics 2011 13, 108-112DOI: (10.1016/j.jmoldx.2010.11.006) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions