Treatment of The Fragile X Syndrome and Why It Should Be Investigated More

The Fragile X Syndrome (FXS) is the number one cause of inherited intellectual disability in males, and the second most common cause of intellectual disability after Down Syndrome. The world prevalence is estimated to be 1:5000-7000 in males and 1:4000-6000 in females [2]. Physical phenotype includes long face, large and protruding ears, hyper extensible finger joints, high-pitched jocular speech, among others; while intellectual disabilities can range from learning disabilities in the context of normal intelligence to severe mental retardation. The syndrome is not curable yet, hence all the available treatments are symptomatic and physical and educational therapy is recommended.

The FXS was first identified in 1943 by Martin and Bell as a type of intellectual disability associated to the X gene [3]. Currently, it is attributed to the functional knock-out or reduced expression of the Fragile X Mental Retardation-1 gene (FMR1), localized in the telomeric region of the X chromosome’s long arm. This transcription deficiency is explained by the increased number of methylated CGG trinucleotide repeats within the untranslated exon 1 of the FMR1 gene (generally, above 200) and the hypermethylation of the CpG islands neighboring the expanded CGG repeats characterizing the presence of the cytogenetic fragile site (FRAXA).

The combination of these factors results in the heterochromatin conformation of the FMR1 promoter region leading to gene silencing and it is referred to as a full mutation (FM). Furthermore, a pre-mutation (PM) occurs whenever the number of repeats ranges from 55-200 and, although the individual is clinically unaffected, a transition to FM is possible. Transition occurs when a parent (rarely the father) carrying the PM variant transmits it to the children and the number of CGG repeats increases above 200 due to the expansion phenomenon.

The most common forms of diagnosis consist in observing physical and mental phenotypes. On the other hand, there are some alternatives available for the molecular diagnosis of FXS including cytogenetic, Southern-blot, PCR, reverse transcription PCR, and immunohistochemical analyses. Those procedures focus on identifying either the expanded CGG repeat, the hypermethylation of the affected regions, or both. Radioactive or non-radioactive southern-blotting with methyl-sensitive and methyl-insensitive restriction enzymes is the most reliable technique for covering the whole spectrum of pre-mutations, full mutations, and mosaicism.

While PCR-based alternatives are capable of determining the length of the CGG repeat in the normal and premutation range, or detecting the methylated FMR1 promoter, or observing absence of gene expression. Additionally, the absence of the respective protein can as well be assessed immunohistochemically with a FMR1-directed antibody. The aforementioned techniques are accurate for the diagnosis of male individuals; however, the diagnosis of female individuals remains a greater challenge. Although, the application of PCR techniques combining repeat-length and methylation analysis can improve the detection of pathological FRAXA variants in females [8].

The hypermethylation of nucleotides on the chromosome X is tightly associated with the expansion of CGG repeats and the disease status. Normal and PM individuals are characterized by an unmethylated FMR1 gene while FM individuals present methylation on the 5’-region surrounding the promoter and on the expanded repeat in the 5’ untranslated region. However, FMR1 alleles with multiple repeats (300-800) can be reactivated in vitro after treatment with demethylating agents. This evidence combined with many reports of unmethylated FM males with full cognitive capabilities and approximately normal levels of FMR1 expression suggest that the disease status is associated to the methylation of the FMR1 promoter rather than the methylation of the expanded repeats or solely CGG repeat number.

One of the most successful approaches investigated for the treatment of FXS involves the inhibition of the glycogen synthase kinase-3 (GSK3). Studies conducted on the mouse model that lacks FMR1 expression report that chronic dietary lithium treatment improves impaired cognition on both adolescent and adult mice and preliminary evidence suggests that lithium might be effective in human patients as well. Furthermore, the administration of different GSK3 inhibitors, such as TDZD-8 or VP0.7, also ameliorated cognitive function. Inhibition of GSK3 enables the optimal establishment of long-term potentiation (LTP) and the rescue of abnormal LTP and/or long-term depression (LTD) regulating hippocampal synaptic plasticity and improving memory and learning in the FXS mice model. [9,10]

More than seven decades have passed since the discovery of the Fragile X Syndrome and this inheritable intellectual disability remains prevalent worldwide. The application of modern molecular biology techniques shed the light on the underlying epigenetic mechanism of the disease and gave rise to new methods for more precise diagnosis. Nevertheless, more investigation is required in order to develop more effective treatments and possible preventive measures for PM parents focused on the epigenetic factor of the disease rather than the symptoms solely.