1. FMR1 Intron 1 Methylation Predicts FMRP Expression in Blood of Female Carriers of Expanded FMR1 Alleles 
David E. Godler, Howard R. Slater, Quang M. Bui, Michele Ono, Freya Gehling, David Francis, David J. Amor, John L. Hopper, Randi Hagerman, Danuta Z. Loesch 
The Journal of Molecular Diagnostics 
Volume 13, Issue 5, Pages (September 2011)
DOI: /j.jmoldx
Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
Examples of raw mass spectra for selected FREE1 and FREE2 CpG sites from spiked samples. The mass difference between the NM and M fragments for the same CpG site is 16 Da because of the presence of an adenosine residue in the place of a guanosine residue. The EpiTYPER-used software identifies peaks acceptable for analysis, which is presented in the form of the MOR.29–31 This ratio represents the relationship between M and NM peaks for the same fragments, which is analogous to total percentage of methylation.26 Herein, we present raw spectra for spiked samples included during each run used to indicate if there were any technical problems between different plates/runs and whether the results were comparable between the runs. Lymphoblast DNA from an HC male was spiked with lymphoblast DNA form a male affected with FXS at 1:0, 2:1, 1:1, 1:2, and 0:1 ratios, corresponding to 0%, 33.3%, 50%, 66.6%, and 100% (top to bottom), respectively, FXS DNA in the sample. The peaks representing fragments from UM CpG units are represented by red lines, whereas the fragments from methylated CpG units are represented by blue lines. A: CpG4 fragments with no silent peaks. B: FREE2 CpG 10, 11, and 12 fragments of the same size, with the third blue peak representing the combined mass of methylation fragments from all three sites (54 Da away from the UM peak). C: FREE1 CpG 10 fragments with a silent peak contribution evident (bottom panel). D: FREE1 CpG 9 fragments with a silent peak contribution evident (bottom panel). The corresponding MORs from these raw spectra are plotted against FXS DNA input (see Supplemental Figures S2 and S3 at MOR indicates methylation output ratio.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Figure 2
The relationship between methylation of specific CpG units within FREE1 and the proportion of FMRP-positive cells in the blood of PM and FM females. MORs for CpG units from 2 to 10 were negatively correlated with the FMRP expression in the cohort of 12 PM and 22 FM females. (Black diamonds indicate FM; white diamonds indicate PM.) Analysis of the MOR was calculated based on the sum of intensities for M/(M + UM signal), as previously described.26 A: CpG1 did not show a significant correlation with FMRP expression. B: CpG2. C: CpG3. D: CpG4. E: CpG5/6. F: CpG8. G: CpG9. H: CpG10. CpG sites 5 and 6 showed fragment sizes that overlapped using MALDI-TOF analysis and were expressed as the mean methylation value, representing two sites presented as CpG unit 5/6. The broken lines represent the methylation range (mean ± SD) for each CpG unit in HC females, which is listed in Table 1.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Figure 3
The relationship between methylation of specific CpG units within FREE2 and the proportion of FMRP-positive cells in the blood of PM and FM females. MORs for CpG units from 1 to 12 were negatively correlated with the FMRP expression in the combined cohort of 12 PM and 22 FM females. (Black diamonds indicate FM; white diamonds indicate PM.) A: CpG1. B: CpG2. C: CpG 6/7. D: CpG 8/9. E: CpG 10 to 12. An analysis of CpG sites 6 and 7 and CpG sites 8 and 9 showed fragment sizes that overlapped using MALDI-TOF analysis and were expressed as the mean CpG unit methylation value of two sites presented as CpG units 6/7 and 8/9, respectively. The fragment sizes for CpG sites 8, 9, and 10 also overlapped and were presented as the CpG unit 10–12 denominations, representing mean methylation across the three sites. The broken lines represent the methylation range (mean ± SD) for each CpG unit in HC females, which is listed in Table 1.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. Figure 4
The correlation between FREE region methylation and FMRP immunostaining in blood of the combined PM and FM cohort of females and between the FMR1 AR and FMRP. A: The relationship between mean methylation across CpG units 2 to 10 of FREE1 and FMRP immunostaining in blood. B: The relationship between mean methylation across CpG units 1 to 12 of FREE2 and FMRP immunostaining in blood. C: The relationship between FMR1 AR, determined using methylation-sensitive (NruI site) SB analysis and FMRP immunostaining in blood. Simple linear regression was performed for A–C on the combined cohort of PM and FM females and on only the FM cohort. Black diamonds indicate FM; white diamonds indicate PM.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions