S. Hussain Askree, Shika Dharamrup, Lawrence N. Hjelm, Bradford Coffee

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Parent-of-Origin Testing for 15q11-q13 Gains by Quantitative DNA Methylation Analysis S. Hussain Askree, Shika Dharamrup, Lawrence N. Hjelm, Bradford Coffee The Journal of Molecular Diagnostics Volume 14, Issue 3, Pages 192-198 (May 2012) DOI: 10.1016/j.jmoldx.2012.01.005 Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Map for SNRPN Southern blot and Q-MSP design. A: Location of the SNRPN gene within the 15q11.2-q13 chromosome segment. B: Map of the 4-kb XbaI restriction fragment that is differentially cut with methylation-sensitive KspI restriction sites within the SNRPN promoter's CpG island. The 4-kb fragment in the unmethylated paternal allele is further cut into 1.1-kb and 2.9-kb fragments. The radiolabeled probe used in Southern blot analysis (not shown) corresponds to sequence within the SNRPN promoter and targets a 1.1-kb band in the unmethylated paternal allele and a 4-kb band in the methylated maternal allele. C: Map of the SNRPN promoter's CpG island, showing the 120-bp sequence amplified in the Q-MSP assay. Green stars indicate CpG locations. Bars indicate DNA-specific probes: FAM-SNRPN-M, methylated (red); VIC-SNRPN-U, unmethylated (blue). The Journal of Molecular Diagnostics 2012 14, 192-198DOI: (10.1016/j.jmoldx.2012.01.005) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 SNRPN DNA sequence with primers and probes used for Q-MSP analysis in testing for PWS and AS. The sequence is shown in three forms: the genomic reference sequence (untreated) and the maternal (methylated) and paternal (unmethylated) sequences, the latter two after sodium bisulfite treatment. The CpG dinucleotides are highlighted in yellow. Primer and probe nucleotides are indicated in underscored bold type. The reverse primer 15q_RealT_R has one CpG within its length (arrow) and is therefore a pool of the two sequences for the two alleles. The sequence of the SNRPN promoter that is amplified with these primers corresponds to genomic position Chr15:22,751,101–22,751,220 (NCBI36/hg18). The Journal of Molecular Diagnostics 2012 14, 192-198DOI: (10.1016/j.jmoldx.2012.01.005) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 A: Methylation-sensitive Southern blot analysis of the SNRPN promoter. Molecular weight marker (M), Lane 1, DNA from a patient with an isodicentric marker chromosome 15; lane 2, DNA from a patient with a 15q11.2-q13 interstitial duplication on the paternally inherited chromosome 15; lane 3, DNA from a patient with a 15q11.2-q13 interstitial duplication on the maternally inherited chromosome 15; lane 4, DNA from an unaffected individual. B: Densitometric analysis of the Southern blot in terms of MI. Iso mar, isodicentric marker; Mat dup, maternal duplication; Neg ctrl, negative control; Pat dup, paternal duplication. The Journal of Molecular Diagnostics 2012 14, 192-198DOI: (10.1016/j.jmoldx.2012.01.005) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Q-MSP analysis of the SNRPN promoter in Prader-Willi syndrome and Angelman syndrome patients. DNA from a negative control (A), from a PWS patient (B), and from an AS patient (C). All samples were assayed in triplicate; representative amplification plots are shown. The Journal of Molecular Diagnostics 2012 14, 192-198DOI: (10.1016/j.jmoldx.2012.01.005) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Titration of bisulfite-treated DNA from unaffected individuals was used to generate standard curves for quantification of methylated and unmethylated SNRPN DNA. Representative amplification plots (left) and corresponding standard curves (right) are shown, used to quantify the amount of methylated (detected by the FAM-labeled probe) and unmethylated (detected by the VIC-labeled probe) SNRPN DNA. The Journal of Molecular Diagnostics 2012 14, 192-198DOI: (10.1016/j.jmoldx.2012.01.005) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Verification for SNRPN Q-MSP. A: Amplification plots from samples with 15q11-q13 gains that were assayed with Southern blot. DNA from an unaffected individua1 (Neg ctrl), DNA from a patient with a 15q11.2-q13 interstitial duplication on the paternally inherited chromosome 15 (Pat dup), DNA from a patient with an interstitial duplication on the maternally inherited chromosome 15 (Mat dup), and DNA from a patient with a supernumerary chromosome 15 [inv dup(15)] that leads to a single copy of the unmethylated allele and triplication of the methylated allele of 15q (Iso mar). B: Q-MSP quantification of SNRPN methylation from the four samples after interpolation of the CT values on a standard curve. The yellow area indicates the normal range (Table 1). The Journal of Molecular Diagnostics 2012 14, 192-198DOI: (10.1016/j.jmoldx.2012.01.005) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions