Laila C. Schenkel, Charles Schwartz, Cindy Skinner, David I

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Clinical Validation of Fragile X Syndrome Screening by DNA Methylation Array Laila C. Schenkel, Charles Schwartz, Cindy Skinner, David I. Rodenhiser, Peter J. Ainsworth, Guillaume Pare, Bekim Sadikovic The Journal of Molecular Diagnostics Volume 18, Issue 6, Pages 834-841 (November 2016) DOI: 10.1016/j.jmoldx.2016.06.005 Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 DNA methylation analysis of FMR1 promoter in FXS patients versus controls. The figures show the methylation levels (0 = 0% methylation, 0.5 = 50% methylation, 1 = 100% methylation) at specific array probes at the FMR1 gene promoter; the CpG island location (gray boxes); and the reference sequence (RefSeq) for 5′ to 3′ strand (+) and for 3′ to 5′ strand (−). A: FMR1 mean methylation levels in males with full mutation (FM; red), mosaic-FM (green), premutation (PM; blue), and controls (black). B: Hypermethylation in FM FXS male patients (red) compared to male controls (black): individual sample profiles. C: Hypermethylation in mosaic-FM male patients (green) compared to male controls (black): individual sample profiles. D: Mean methylation levels in females with FMR1 FM allele (red) and PM (blue) compared to female controls (black). E: Methylation levels in FM carrier females (red) compared to female controls (black): individual sample profiles. The Journal of Molecular Diagnostics 2016 18, 834-841DOI: (10.1016/j.jmoldx.2016.06.005) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 A: Mean methylation for all 13 probes at FMR1 gene promoter in male controls, premutation males (PM), mosaic–full mutation (FM), and FM FXS patients. A methylation value cutoff of 0.2 enables accurate identification of FXS patients. B: Mean methylation for all 13 probes at FMR1 gene promoter in female controls, PM, and FM female carriers. ∗∗∗∗P < 0.0001 (methylation level is significantly different). The Journal of Molecular Diagnostics 2016 18, 834-841DOI: (10.1016/j.jmoldx.2016.06.005) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Differential DNA methylation analysis using genome-wide methylation array. The figures show the methylation levels (0 = 0% methylation, 0.5 = 50% methylation, 1 = 100% methylation); the CpG island location (gray boxes); and the reference sequence (RefSeq) for 5′ to 3′ strand (+) and for 3′ to 5′ strand (−). A: DNA methylation pattern at SNRPN locus on chromosome 15 in patients with Prader-Willi syndrome (red) and Angelman syndrome (blue). B: Episignature in patients with Sotos syndrome; two patients (blue and red) show similar DNA methylation changes at two loci on chromosome 6 when compared with controls (black). Both genes have recently been described as part of the unique Sotos episignature.32 The Journal of Molecular Diagnostics 2016 18, 834-841DOI: (10.1016/j.jmoldx.2016.06.005) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S1 There is no correlation between CGG repeat size and FMR1 promoter methylation levels in full mutation FXS patients. Mean methylation at FMR1 gene promoter (all 13 probes) (y axis) and CGG expansion size in full mutation FXS patients (x axis); R2 = 0.0083. The Journal of Molecular Diagnostics 2016 18, 834-841DOI: (10.1016/j.jmoldx.2016.06.005) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S2 Single-probe methylation patterns differ significantly between male controls, full mutation (FM) FXS patients, and mosaic FXS patients. Thirteen probes spanning the 1482-bp FMR1 promoter region were evaluated. The graphs show median, maximum, and minimum methylation values for each of the probes in male controls, FM patients, and mosaic patients. Circles represent individual samples. Probe locations on FMR1 promoter region are shown on bottom image. Overall, male controls are hypomethylated across the FMR1 promoter region; mosaic FXS males show a higher range of methylation compared to controls (P < 0.01 for all 13 probes); and full mutation FXS males show even higher levels of methylation (hypermethylation) at each of the FMR1 promoter region probes (P < 0.01 compared with controls, and P < 0.01 compared with mosaic FXS males). The Journal of Molecular Diagnostics 2016 18, 834-841DOI: (10.1016/j.jmoldx.2016.06.005) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions