Tissue mosaicism, FMR1 expression and intellectual functioning in males with fragile X syndrome

Fragile X syndrome (FXS) is caused by hypermethylation of the FMR1 promoter due to the full mutation expansion (full mutation [FM]: CGG ≥ 200 repeats) and silencing of FMR1. Assessment of mosaicism for active-unmethylated alleles has prognostic utility. This study examined relationships between FMR1 methylation in different tissues with FMR1 messenger ribonucleic acid (mRNA) and intellectual functioning in 87 males with FXS (1.89-43.17 years of age). Methylation sensitive Southern blot (mSB) and Methylation Specific-Quantitative Melt Aanalysis (MS-QMA) were used to examine FMR1 methylation. FMR1 mRNA levels in blood showed strong relationships with FMR1 methylation assessed using MS-QMA in blood (n = 68; R²  = 0.597; p = 1.4 × 10^-10 ) and buccal epithelial cells (BEC) (n = 62; R²  = 0.24; p = 0.003), with these measures also showing relationships with intellectual functioning scores (p < 0.01). However, these relationships were not as strong for mSB, with ~40% of males with only FM alleles that were 100% methylated and non-mosaic by mSB, showing methylation mosaicism by MS-QMA. This was confirmed through presence of detectable levels of FMR1 mRNA in blood. In summary, FMR1 methylation levels in blood and BEC examined by MS-QMA were significantly associated with FMR1 mRNA levels and intellectual functioning in males with FXS. These relationships were not as strong for mSB, which underestimated prevalence of mosaicism.