Methylation of novel markers of fragile X alleles is inversely correlated with FMRP expression and FMR1 activation ratio

Abstract

The fragile X syndrome (FXS) is caused by silencing of the fragile X mental retardation gene (FMR1) and the absence of its product, fragile X mental retardation protein (FMRP), resulting from CpG island methylation associated with large CGG repeat expansions (more than 200) termed full mutation (FM). We have identified a number of novel epigenetic markers for FXS using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), naming the most informative fragile X-related epigenetic element 1 (FREE1) and 2 (FREE2). Methylation of both regions was correlated with that of the FMR1 CpG island detected using Southern blot (FREE1 R = 0.97; P < 0.00001, n = 23 and FREE2 R = 0.93; P < 0.00001, n = 23) and negatively correlated with lymphocyte expression of FMRP (FREE1 R = -0.62; P = 0.01, n = 15 and FREE2 R = -0.55; P = 0.03, n = 15) in blood of partially methylated 'high functioning' FM males. In blood of FM carrier females, methylation of both markers was inversely correlated with the FMR1 activation ratio (FREE1 R = -0.93; P < 0.0001, n = 12 and FREE2 R = -0.95; P < 0.0001, n = 9). In a sample set of 49 controls, 18 grey zone (GZ 40-54 repeats), 22 premutation (PM 55-170 repeats) and 22 (affected) FXS subjects, the FREE1 methylation pattern was consistent between blood and chorionic villi as a marker of methylated FM alleles and could be used to differentiate FXS males and females from controls, as well as from carriers of GZ/PM alleles, but not between GZ and PM alleles and controls. Considering its high-throughput and specificity for pathogenic FM alleles, low cost and minimal DNA requirements, FREE MALDI-TOF MS offers a unique tool in FXS diagnostics and newborn population screening.

Fragile X–related element 2 methylation analysis may provide a suitable option for inclusion of fragile X syndrome and/or sex chromosome aneuploidy into newborn screening: a technical validation study

“…11, methylation analysis was performed on eight DNA samples on 14 different occasions over a 7-month period by three different operators, with skill sets ranging from experienced to novice. Three of these samples were made by spiking DNA from a healthy male with DNA from an FXS-affected male at different ratios (Figure 1a).…”

“…This punch should be converted in two bisulfite reactions, with each conversion used for one PCR/mass cleave reaction, and the mean of the two separate reaction representing the final value. It is also important to note that the technical limitation of this approach is that it will not detect a difference in methylation less that 10% of the true value, which is analogous to the contribution of the combined technical variation for FREE1 analysis 11 ; and this limitation should be used when interpreting differences in biological variation.…”

“…11 Methylation analysis for each sample was performed in duplicate (unless otherwise stated), giving two separate methylation output ratios (MORs), reflecting technical variation resulting from bisulfite conversion, PCR, and mass cleave reactions.…”

“…[10][11][12][13] This test also has the potential to be used for detection of sex chromosome aneuploidy.…”

“…22,23 The FREE2 blood test identifies cognitively impaired FM males and females with specificity and sensitivity approaching 100%, but cannot distinguish between small-expansion carriers and healthy controls or high-functioning males with unmethylated FM alleles. [11][12][13] This is highly advantageous for newborn, infant, or early childhood population screening because the test potentially detects only those who would directly benefit from early diagnosis. 24 In this study, we performed technical validation for the FREE2 analysis in detection of FXS alleles in archival newborn and adult dried blood spots and venous blood DNA.…”