Relationships between age and epi-genotype of the FMR1 exon 1/intron 1 boundary are consistent with non-random X-chromosome inactivation in FM individuals, with the selection for the unmethylated state being most significant between birth and puberty

Abstract

Methylation of the fragile X-related epigenetic element 2 (FREE2) located on the exon 1/intron 1 boundary of the FMR1 gene is related to FMRP expression and cognitive impairment in full mutation (FM; CGG>200) individuals. We examined the relationship between age, the size of the FMR1 CGG expansion and the methylation output ratio (MOR) at 12 CpG sites proximal to the exon 1/intron 1 boundary using FREE2 MALDI-TOF MS. The patient cohort included 119 males and 368 females, i.e. 121 healthy controls (CGG<40), 176 premutation (CGG 55-170) and 190 FM (CGG 213-2000). For all CpG units examined, FM males showed a significantly elevated MOR compared with that in hypermethylated FM females. In FM males the MOR for most CpG units significantly positively correlated with both age and CGG size (P< 0.05). In FM females the skewing towards the unmethylated state was significant for half of the units between birth and puberty (P < 0.05). The methylation status of intron 1 CpG10-12 that was most significantly related to cognitive impairment in our earlier study, did not change significantly with age in FM females. These results challenge the concept of fragile X syndrome (FXS)-related methylation being static over time, and suggest that due to the preference for the unmethylated state in FM females, X-inactivation at this locus is not random. The findings also highlight that the prognostic value of FXS methylation testing is not uniform between all CpG sites, and thus may need to be evaluated on a site-by-site basis.

Figure 1.

Comparison between CGG size (x-axis) and methylation output ratio (y-axis) within the FREE2 region. (A) Representation of the intron and exon regions 3′ of the FMR1 CGG expansion (sequence numbering from GenBank L29074 L38501) in relation to FMR1 and ASFMR1 transcription start sites (the broken lines indicated spliced out regions), FREE2 and the FMR1 CpG island and methylation-sensitive restriction sites (NruI, EagI and BssHII) analysed using routine fragile X Southern blot testing. A CGG repeat is located within the 5’ (UTR) of the FMR1 gene. ASFMR1 spans the CGG expansion in the antisense direction and is also regulated by another promoter located in the exon 2 of FMR1. The FREE2 region is located downstream of the CGG expansion, with CpG1 and 2 located within the 3′ end of FMR1 exon 1; CpG6/7, 8/9 and 10–12 located within the 5′ end of FMR1 intron 1. (B) 119 males with CGG size ranging between 22 and 2000 repeats. (C) 368 females with CGG size ranging between 21 and 1500 repeats. Note: PM/FM mosaic individuals and ‘high functioning’ unmethylated FM males as determined by methylation-sensitive Southern blot were not included. For females only the size of the smallest size expanded allele is presented on the x-axis. If a FM allele was identified as a smear; the lowest CGG size expanded allele was presented on the x-axis for both FM males and females. The horizontal-broken line represents the maximum MOR value for the control group with CGG<40, with this value indicated above this line, with an exception of CpG6/7 where one control outlier at the MOR of 0.63 was not considered as the maximum control value. The perpendicular-broken line represents the minimum MOR value between 100 and 300 CGG repeats which is above the maximum value of the control group.

Figure 2.

FREE2 methylation comparisons between FM males and females with the methylation output ratio above the maximum MOR of the control group. CpG1 and 2 are located within the 3′ end of FMR1 exon 1; CpG6/7, 8/9 and 10–12 are located within the 5′ end of FMR1 intron 1. Note: All comparison showed P < 0.001. CpG1—open circles; CpG2—open squares; CpG6/7—closed squares; CpG8/9—closed triangles; CpG10–12—open diamonds.

Figure 3.

Segmented linear regression model comparison between age (x-axis) and methylation output ratio (y-axis) within the FREE2 region of 104 FM females with CGG size ranging between 21 and 1500 repeats. The perpendicular-broken line represents the ‘break point’ age estimated using the segmented linear regression model as detailed in Table 3. From birth to the ‘break point’ age there was significant inverse relationship with MOR for CpG 1, 2 and 6/7, but not CpG8/9 and 10–12. At the ages past the ‘break point’ there was no significant relationship with MOR observed for any of the CpG units. The horizontal-broken line represents the maximum MOR value for the control group with CGG<40, with this value indicated next to this line, with an exception of CpG6/7 where one control outlier at the MOR of 0.63 was not considered as the maximum control value. Note: PM/FM mosaic individuals and FM methylation mosaics as determined by methylation-sensitive Southern blot were not included.