Figure 3

The percentage of methylated imprinting center of the Prader-Willi locus (PWS-IC) shows a positive correlation with copy number at the 15q locus. (a) Example of methylation-sensitive high-resolution melting-curve analysis showing > 80% methylated PWS-IC for duplication of 15q11-q13 (dup15q) sample 7041 (3:1 expected genetic ratio of the number of maternal copies of 15q11-q13 to paternal copies (M:P)) and sample 7014 (5:1 expected M:P ratio) (blue curves). All control and autism samples showed, on average, a 62% methylated PWS-IC (sample 6184 autism (orange) and sample 1649 control (green) here). The Prader-Willi syndrome uniparental disomy (PWS-UPD) (yellow) and the Angelman syndrome (AS) deletion (dark red) are shown as indicators of completely unmethylated (AS del) or completely methylated (PWS-UPD) signals from the PWS-IC. The blue arrows show the determination of percentage methylation from the percentage relative signal y-axis. (b) This graph presents the normalized methylation ratio (M:P) shown in Figure 1 grouped by genotype. Note that both control and autism samples cluster tightly at the 1:1 ratio in all samples. There was a single interstitial duplication sample (predicted 2:1 ratio) and a single complex isodicentric 15q (idic15) duplication sample (predicted 5:1 ratio), though the majority of cases examined were idic15q with four copies of the locus (predicted 3:1 ratio). Although somewhat variable, the mean ratio for the Dup 3:1 samples was approximately 3:1 and significantly different from both controls (P = 0.0037) and autism cases (P = 0.0035) by t-test using Welch's correction. The individual Dup 2:1 and Dup 5:1 samples showed higher and lower than predicted ratios, respectively. Error bars represent SEM. There was a positive correlation between the percentage methylation of the PWS-IC and the number of copies of the 15q region, which contains the PWS-IC, on the basis of simple regression analysis (P < 0.001).