Standard laboratory testing for FXS falls into the following categories. The first category applies to diagnostic testing of individuals with intellectual disability, in which a positive result leads to testing of other family members, in whom the risk of carrier status is high. The second category applies to pre-natal testing in known carrier pregnancies. The third category is the population screening of newborns. Earlier treatment intervention, identification of probands pointing to high-risk relatives and provision of reproductive counselling are strong arguments in favour of newborn screening. One major impediment is the limited suitability of current test methods, which are a combination sizing of small and large CGG repeat expansions by PCR and Southern blot testing, respectively (23
). The PCR-based assays only target CGG size and do not provide information on the state of the FMR1 promoter methylation (24
). This shortcoming necessitates further testing using methylation-sensitive Southern blot. However, the use of Southern blot for any type of large-scale testing is primarily restricted by low throughput, cost and DNA quality and quantity limitations. Importantly, PCR testing detects GZ and PM carriers who are highly prevalent in the general population (for males one in approximately 30 for GZ and one in approximately 700 for PM carriers and for females one in approximately 15 for GZ and 1 in approximately 250 for PM carriers) (34
). These small expansions do not cause FXS, but PM carriers have a high risk of transmitting them in an expanded form. Furthermore, both PM and GZ alleles have been associated with elevated FMR1 mRNA (18
) and related to increased risks of developing FXPOI, and PM alleles have also been linked to FXTAS (5
). Thus, a test that detects GZ and PM alleles may inadvertently turn a screen for FXS into a predictive assay for a late onset disorder raising unavoidable ethical issues. Furthermore, the counselling costs generated by finding these alleles are prohibitive and of questionable value. There has, therefore, been no ethically acceptable, simple, accurate and inexpensive test available for FXS population screening.
These problems might now be overcome by a novel MALDI-TOF MS test for methylated markers of FXS described in this study. The method is advantageous over most other MS-PCR-based assays (37
) and enzyme-based methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA) methods (39
) developed to examine the methylation of the classical FMR1 CpG island, as MALDI-TOF MS can be used to rapidly examine large stretches of DNA for methylation. In contrast, most PCR or MS-MLPA and enzyme-based MS-MLPA methods are restricted to a few sites that are less biologically significant and/or more heavily affected by skewed X-inactivation (37
). MALDI-TOF MS described in this study requires ~100-fold less DNA quantity than Southern blot and ~10-fold less DNA quantity than the MS-PCR established for FXS testing in males (38
). It is less sensitive to DNA quality issues than Southern blot (as it was used to identify FM/FXS samples that failed when analysed via Southern blot; unpublished data), is far more rapid (<2 days versus 1–2 weeks for Southern) and is less expensive. Furthermore, MALDI-TOF MS has been used previously to assess methylation from both freshly prepared and archival dried blood spots (40
Using this methodology, we identified two novel regions that were hypermethylated in FXS individuals, but unmethylated in carriers of smaller expansions. We have named the region 5′ of the promoter FREE1 and the region 3′ of the CGG expansion, largely located within intron 1 of the FMR1, FREE2. The two regions were of similar size and CG content, with the CpG unit density approximately one-third of that found in the 52 unit classical CpG island described previously (27
). Based on the GC content, the observed/expected CpG ratio and the length of the fragment, we propose that the FREE1 region should be considered as an extension of the classical CpG island (27
). In contrast, we could not classify FREE2 as a part of the island, as the regions are separated by the CGG expansion. When considered separately, the FREE2 observed/expected CpG ratio is significantly lower than that used for CpG island classification (32
). However, due to its orthologous conservation (Supplementary Material, Table S2
), it can be considered as a CG cluster (41
). The presence of a number of conserved putative GATA binding sites within this region further suggests that FREE2 may be involved in transcriptional regulation (42
In contrast to FREE2, the human FREE1 sequence is only homologous to that of the common chimp. This lack of conservation may explain why FREE1 was not previously considered functionally important in the regulation of FMR1 transcriptional activity (28
) and was not included as part of the FMR1 CpG island (27
). However, lack of sequence conservation does not exclude functional importance for long non-coding RNA species, particularly for those acting in cis
) and in trans
). Since the FREE1 region falls at the 5′ end of FMR4—a long non-coding RNA (47
)—it may also have functional significance despite the absence of interspecies homology. This is supported by our sequence analysis of FREE1, in which we have localized a number of putative transcription factor binding sites, suggesting the potential importance of this region in transcriptional regulation. Of particular interest is the (CAAAC)n repetitive element that encompasses more than 11 putative SRY binding sites and the GATA1/2-SRY-Ik2-c-Ets transcription factor binding region in the 5′ portion of FREE1.
Since we found that the mean methylation of the FREE1 and FREE2 regions on either side of the previously described FMR1 promoter (27
) was closely related, we propose that these markers are extensions of the same FMR1 regulatory region. This is further supported by our observations of FREE1 and FREE2 hypermethylation in the FXS cell lines with silenced FMR1 transcription and FMRP expression (Supplementary Material, Fig. S1
). It is also of interest that one of these cell lines was taken from a female carrying FM with completely skewed X-inactivation, implying that in all cells of this patient the methylated FM was on the active X. This is consistent with the fact that this 37-year-old female was the most retarded carrier in her family (IQ<30; pedigree no. II.5), with the most severe emotional and physical abnormalities (48
). It is fascinating that although this female had methylation of most CpG units within FREE1, FREE2 and the classical CpG island approaching 100%, the regions adjacent to FREE1 amplicons 3 and 4 (that are largely hypermethylated in controls) were ~50% methylated in this female. Since a similar pattern was observed in the FXS cell lines from the affected males, this may suggest that in FXS, methylation shifts from the upstream regions (detected by amplicons 3 and 4) to FREE1, and this may have implications for transcription from the FMR1 locus and the FXS phenotype.
The potential regulatory involvement of the FREE1 and FREE2 regions is further supported by our observations of a significant positive association between methylation status of the two regions and that of the classical CpG island determined by Southern blot analysis in blood of high functioning males, including mosaic individuals. In addition, we have observed a significant inverse correlation between FREE1 and FREE2 and the number of FMRP-positive lymphocytes and the FMR1 activation ratio in blood of high functioning males and of the females with variable FMR1 activation ratio that carried FM alleles. It is of interest to note that mean FREE1 methylation was more closely related to FMRP expression in blood than methylation of the NruI site examined using Southern blot, emphasizing its potential importance as a novel marker in FXS diagnostics.
In a larger sample set, the FREE1 methylation pattern was generally consistent between blood, amniocytes and chorionic villus as a marker of methylated FM alleles and X-inactivation and could be used to differentiate FXS males and females from controls, as well as from carriers of GZ and high PM alleles (expanded up to 170 repeats). However, the FREE1 methylation analysis could not differentiate between GZ and PM alleles and controls—a technical drawback that could also be a major advantage of the MALDI-TOF MS over the existing methodologies in a newborn screening programme.
In terms of potential pre-natal applications, it is important to note that in CVS of FM carriers, the methylation pattern is not necessarily consistent between all CpG sites within the classical CpG island, meaning that the analysis of a specific site using Southern blot may not necessarily reflect the methylation of a different restriction site and the CpG island as a whole. This is primarily based on reports of discordance in the methylation status of a specific site (BssHII, co-localized to footprint IV of the CpG island) in a proportion of samples between male foetal tissues and the corresponding CVS taken at 8 weeks of gestation (49
), and lack of methylation at the FMR1 locus related to X-inactivation in CVS but not in foetal cells (17
). In contrast, Devys et al
) have reported that hypermethylation of a different site (EagI located 10 bp upstream of footprint IV) in CVS of FM carrier males occurred as early as 10 weeks of gestation and was strikingly consistent between CVS, cord blood and different foetal tissues, as well as the clinical FXS phenotype after birth (17
). The same may apply to different CpG sites within FREE1 analysed in chorionic villi taken between 10 and 20 weeks of gestation; however, because our analysis looks at methylation pattern across nine sites, instead of one (which is the case for Southern blot), FREE1 analysis may provide a better alternative for methylation analysis in pre-natal samples. However, before our technique is used in pre-natal diagnosis, a much larger sample size should be analysed for the consistency of these methylation patterns in CVS and amniocytes of FM carriers.
Another potential limitation is that the FREE analysis may not differentiate a proportion of FM carrier females from controls due to severely skewed inactivation, favouring the normal allele on the active X. These FM females may be as common as 30% of all FM female carriers (21
) and are potentially indistinguishable from control females when using methylation or FMRP analysis, without complementary CGG-based tests (52
). These females are also phenotypically relatively normal (54
). However, this limitation can be overcome if instead of Southern blot our tests are used in combination with CGG-based analysis in targeted FXS diagnostics.
Furthermore, although the inability to detect female FM carriers with completely active normal allele may be a limitation from the viewpoint of calculating reproductive risks in adult population, it is far less relevant or may in fact be an advantage for newborn screening, where the focus is on early detection of abnormal phenotype and use of this information for improvement in the prognosis for FXS children through early treatment intervention (55
). In addition, if our tests fail to identify girls who may carry FM alleles early in life, but otherwise may be clinically normal, the lack of this information can potentially avoid stigma related to ethical issues.
In summary, we have shown that the FREE MALDI-TOF MS can accurately identify all carriers of pathogenic methylated CGG repeat expansion mutations of FMR1, while addressing all the main limitations of the existing PCR-based FXS tests, associated with differentiation of unmethylated large PM from methylated FM alleles from males and females as well as detection of unmethylated and/or partially methylated FM alleles (23
). Although applications of this technology in fragile X-related disorders have not been characterized until now, if used in combination with previously described PCR-based analyses that can differentiate between normal size, GZ and PM alleles (24
) in FXS molecular diagnostics, the FREE MOLDI-TOF MS approach can replace Southern blot testing altogether. In contrast, if used alone, the novel test cannot differentiate between GZ and PM and normal range alleles. This avoids ethical issues and high counselling costs associated with the detection of prevalent GZ and PM alleles if used for population screening. Thus, due to its ability to rapidly examine large stretches of DNA for methylation with minimal DNA quantity and quality requirements and at low cost, the FREE MALDI-TOF MS analysis can be added to the existing set of conditions currently tested for in newborns. Early detection through newborn screening may lead to significant improvement in the prognosis for FXS children through early treatment intervention (55
). It may also facilitate detection, through family cascade testing, of other affected or normal transmitting carriers, which can be provided with counselling regarding the ongoing reproductive risks of recurrence for this major cause of intellectual disability.