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.

Figure 1.

Overview of methylation analysis using MADLI-TOF MS/Sequenom system. (A) The protocol is based on in vitro transcription of bisulphate-converted DNA, PCR, base-specific cleavage and fragmentation analysis. (B) An example of a spectrum of fragments from different CpG sites: NM and M fragments for the same CpG site are separated by 16 Da in mass due to the presence of an adenine instead of guanine. The intensity ratio of the cleaved products provides quantitative methylation estimates for CpG sites within a target region. S represents silent peaks, and fragments of unknown origin were not taken into consideration if their size did not overlap with the fragments of interest.

Figure 2.

Promoter region on the FMR1 gene (sequence numbering from GenBank L29074 L38501) and the adjacent 5′ and 3′ loci. Primers used for MALDI-TOF methylation analysis targeted five regions at the Xq27.3 locus designated as amplicons 1–5 (colour coded). Individual CpG sites within each amplicon are numbered accordingly. The 5′ end of the FMR1 promoter is indicated by ≫ in red (28). The 3′ end of the FMR1 promoter is indicated by ≪ in red (62). The 5′ end of the putative FMR1/ASFMR1 promoter is indicated by > in red, and its 3′ end is indicated by < (64). Prominent transcription factor binding sites and methylation-sensitive restriction enzyme recognition sites are indicated in capital font and are listed/identified in Table 2. The Inr-like transcription start sites are indicated by numbering in red (I–III) embedded within the sequence (65). The main FMR1 transcription start site is indicated by * in red (28,65). The (CGG)n expansion is indicated by red italics.

Figure 3.

The methylation pattern variation at the Xq27.3 locus between controls and FXS individuals. The methylation of individual CpGs was analysed in the 1.766 kb region 5′ and 3′ of the CGG expansion using five MALDI-TOF MS assays (Table 1). DNA from lymphoblasts of (A) controls (n = 4) and (B) FXS patients (n = 3). Methylation output ratios were obtained for T cleavage reactions for most CpG units presented, except for methylation of CpG 7 (underlined and in bold) of amplicon 5, which was obtained from a C-cleavage reaction. * represents missing values, / represents a mean output between two CpG units, - represents a mean output between >2 CpG units. The mean methylation output values represent CpG units with fragments of a similar size that could not be distinguished after the mass cleave reaction. The raw data are presented in Supplementary Material, Table S3.

Figure 4.

Relationship of FREE1 and FREE2 methylation with FMRP levels and FMR1 activation ratio in blood samples from high functioning males and FM carrier females. The relationship between FMRP-positive cell counts in blood smears and Southern blot methylation analyses, FMR1 mRNA levels and standardized IQ for some of these patients was previously described by Tassone et al. (33). (A) The mean methylation across FREE1 (CpG units 2–10) and FREE2 was positively correlated with Southern blot analysis and negatively correlated with the FMRP expression in 21 high functioning males. (B) The mean methylation across FREE1 (CpG units 2–10) and FREE2 was negatively correlated with the FMR1 activation ratio determined using Southern blot analysis in 12 FM and 9 FM carrier females, respectively. The broken line represents the mean methylation output ratio in 11 control females analysed within the same run—baseline ± SD (FREE1 0.43 ± 0.04; FREE2 0.25 ± 0.025; Southern blot 0.39 ± 0.045). The raw data are presented in Supplementary Material, Table S3.

Figure 5.

Comparison of FREE1 methylation in blood and CVS samples. FREE1 was hypermethylated in all FXS subjects, but not in controls or small allele carriers in blood of males (A) and females (B). (C) FREE1 methylation in male pre-natal samples could also differentiate between methylated FM allele carriers and controls (HC). (A) Male blood of HC (n = 15), GZ (n = 16), PM (n = 14) and FM with FXS (n = 9); (B) female blood of HC (n = 23), GZ (n = 2), PM (n = 8) and FM with FXS (n = 5) and (C) male amniocytes of HC (n = 1), CVS of HC (n = 10) and FM (n = 8). The error bars represent standard error. FXS or FM carrier compared with HC: ^***P < 0.001 and ^**P < 0.05; FXS or FM carrier compared with GZ: ^○○○P < 0.001 and ^○○P < 0.05; FXS or FM carrier compared with PM: ^♦♦♦P < 0.001 and ^♦♦P < 0.05 and PM carrier compared with HC: ^###P < 0.001 and ^##P < 0.05. The raw data are presented in Supplementary Material, Table S3.