Fragile X Syndrome

Fragile X syndrome

Fragile X syndrome, a genetic disorder also referred to as Martin-Bell syndrome, was initially elucidated in 1943 by Martin and Bell as a form of intellectual disability or ID characterized by an X-linked pattern of inheritance. This syndrome represents the most prevalent hereditary cause of varying degrees of cognitive impairment, ranging from mild to severe and serves as the primary monogenic etiology for autism spectrum disorder (ASD). Approximately half of the individuals affected by this syndrome exhibit X-linked intellectual disability, rendering it the leading cause of mental disorder subsequent to trisomy 21. Those afflicted with this condition face a compilation of behavioral, cognitive, neurological and physical challenges. The phenotypic manifestations of Fragile X syndrome encompass attention deficit hyperactivity disorder (ADHD), autism (ASD), apathy and sleep disturbances. Consequently, these patients exhibit an elevated susceptibility to social anxiety, seclusion, impaired communication, abnormal reactions to sensory stimuli, self-inflicted harm, hyperactivity and aggression.

Fragile X syndrome symptoms

Most infants affected by fragile X syndrome exhibit no apparent clinical symptoms at birth and the majority of their physical attributes (such as stature, weight) fall within the range of normal values. however, during early childhood the affected individuals’ physical attributes and growth including growth retardation, Flat feet, elongated facial structure and prominent ears become evident. A challenge in diagnosing fragile X syndrome lies in the delayed onset of clinical symptoms until late childhood, which often leads families to overlook abnormal indications in their offspring and postpone seeking medical attention. Pubertal macroorchidism, characterized by enlarged testicles is an additional feature observed in males affected by fragile X syndrome.

It is estimated that approximately 30-60% of individuals with fragile X syndrome also have autism. In males, complete occurrence of the mutation is associated with complete penetrance and likely presentation of disease symptoms. Conversely, in females, even with complete mutation penetrance is approximately 50% and the severity of clinical symptoms varies from mild to severe. In general, individuals with fragile X syndrome commonly present with certain signs and clinical symptoms, a few of which include:

A large forehead
Large ears with soft cartilage
Dental crowding
Palate with a high arch
Strabismus
Hypotonia
Seizures
Pes planus
Presence of long cracks in the palms
Speech disorders
Inguinal hernia

Fragile X syndrome prevalence

The incidence rate of this disease is approximately one case per 4000 births in males and one case per 8000 births in females. It has been estimated that over 1 in 130 to 250 females are carriers of this disease, while the corresponding number for males is roughly 1 in every 250 to 800 individuals. however, the prevalence of carriers varies across different populations.

Other names for fragile X syndrome

X-linked mental retardation and macroorchidism
Marker X syndrome
FRAXA syndrome
Fra(X) syndrome
Martin-Bell syndrome
FXS

The cause of fragile X syndrome and its inheritance pattern

The primary cause of fragile X syndrome is the mutation in the fragile X mental retardation gene 1 (FMR1) with chromosomal position Xq27.3. In 99% of cases, the mutation associated with this disease entails an expansion and amplification of the CGG three-nucleotide repeats (known as dynamic mutations) within the 5’UTR region of the FMR1 gene. This leads to the methylation and subsequent silencing of the gene, resulting in the absence of the FMRP protein product. Functionally, FMRP plays a crucial role in various aspects of neuronal development and functioning. The region containing the CGG repeats within FMR1 is highly polymorphic and can be classified into four distinct allelic groups:

Normal alleles, characterized by 6 to 44 repetitions with the most prevalent normal alleles in the population typically exhibiting 29 and 30 repetitions within their FMR1 gene.
Intermediate alleles, displaying 45 to 54 repetitions.
Permutated alleles (PM) consist of CGG repeats ranging from 55 to 200. This class of alleles poses a high risk of converting to full mutations (FM) through the occurrence of errors during maternal meiosis. PM alleles generally exhibit increased expression and are associated with related disorders such as fragile X-associated tremor and ataxia syndrome (FXTAS) and fragile X-associated primary ovarian insufficiency (FXPOI). Conversely, FM alleles undergo methylation and silencing, resulting in the absence of FMRP protein.
In the case of full mutation (FM), the number of repetitions exceeds 200. This is accompanied by methylation of cytosine within the CGG and CpG Island repetitive sequences in the upstream region of the promoter. These epigenetic modifications often coincide with histone alterations that are incompatible with transcription, thereby inhibiting the translation of FMRP protein.

FMR1 gene structure

The FMR1 gene consists of 17 exons and spans a region of approximately 38 kilobases (kb). The production of messenger RNA (mRNA) by this gene results in a transcript that is approximately 4.4 kb in length. Through alternative splicing, this mRNA can generate 12 distinct isoforms of the FMRP protein. The molecular weight of these proteins ranges from 70 to 80 kDa. The overall structure of the FMR1 gene encompasses several key components, including a 3′ to 5′ upstream methylated region, a promoter region, CGG repeats and a coding region. In the normal state, the methylated region is located approximately 650 to 800 nucleotides upstream of the CGG sequence. However, in fragile X syndrome alleles this CGG sequence becomes indiscernible. The border region of methylation contains binding sites for various nuclear proteins, including CTCF (CCCTC-Binding Factor), which is a potential transcriptional regulator of this site.

The binding of CTCF likely serves to prevent the spread of methylation towards the promoter region of the FMR1 gene. Recent research has implicated the CTCF protein as a possible regulator of FMR1 expression, potentially exerting its influence through the formation of chromatin rings. The promoter region of the FMR1 gene contains approximately 56 CpG islands and three initiator-like sequences (InR) that are located around 130 nucleotides upstream of the CGG sequence. Transcription of the gene is initiated from one of these three regions, and the size of the CGG repeat may serve as a downstream enhancer/modulator of transcription. As the number of CGG repeats increases, transcription initiation tends to occur at higher regions. These CGG polymorphic repeats are situated in the 5′ untranslated region (UTR) of exon 1. Studies have shown that, on average for every 9 to 10 CGG trinucleotide repeats, there are one or two AGG sequence repeats. The presence of these sequences contributes to the stability of the CGG repeats during DNA replication, thereby reducing the risk of a conversion from a premutation (PM) to a full mutation (FM).

Dynamic mutations involving the expansion of three nucleotide repeats, such as CGG, are classified as loss-of-function mutations. The number of CGG repeats tends to increase over time and across generations, leading to a greater number of affected individuals. This phenomenon is commonly referred to as the Sherman paradox.

FMRP protein

FMRP is an RNA-binding protein that binds to ribosomes, playing a central role in brain development. Additionally, this particular protein is responsible for regulating the translation process of specific messenger RNAs (mRNAs) that are directly involved in the creation of neuronal synapses. Consequently, the gene product of FMR1 plays a significant part in the maturation, adaptability and overall function of synapses. In rare cases, fragile X syndrome arises due to deletions, point mutations and other mutations that occur within the FMR1 gene. The FMRP protein also serves other functions within the cell, one of which is the regulation of intracellular transfers. Within the structure of FMRP, there exists an NLS (Nuclear localization signal) and a NES (Nuclear Export signal), both of which facilitate the movement of the protein between the nucleus and the cytoplasm.

Only a small fraction, approximately 4% of the FMRP protein is located within the nucleus while the majority is situated in the cytoplasm. FMRP is capable of forming a homodimer structure and engaging in interactions with numerous cytoplasmic and nuclear proteins that are involved in mRNA metabolism, such as RNAi and RNA editing. The N-terminal and central regions of FMRP also play a vital role in the remodeling of the cytoskeleton. The function and interactions of this protein are regulated through post-translational modifications, specifically the phosphorylation of amino acids 483 and 521.

Fragile X syndrome diagnosis

Since the discovery of the FMR1 gene in 1991, significant advancements have been made in the diagnosis of FXS. Prior to the identification of this gene, disease diagnosis relied on cell culture and cytogenetic analysis to observe the fragile region of the X chromosome. However, due to the limited visibility of the fragile area in only a small percentage of cells, particularly in women, many affected individuals could not be accurately identified using this method. Consequently, researchers in this field have dedicated the last century to seeking and developing more precise and sensitive techniques based on molecular and PCR-based approaches to detect FXS.

Presently, the diagnosis of FXS primarily relies on measuring CGG repeats and examining the methylation status of the FMR1 gene utilizing molecular techniques based on fluorescent PCR. These techniques are founded upon the utilization of labeled primers and probes. Fluorescent PCR methods, such as Real-Time PCR and GF-PCR, are extensively employed in disease diagnosis today. Generally, the gold standard methods for diagnosing FXS encompass a combined PCR approach to quantify the quantity of CGG repeats and southern blot analysis to investigate larger alleles and determine methylation status. Although the southern blot method is time-consuming, expensive and necessitates a substantial amount of genomic DNA compared to PCR, it possesses the capability to detect large alleles that are challenging to identify via PCR.

Additionally, this technique provides insight into the methylation status of the allele under investigation. Conventional PCR is incapable of detecting CGG repeats in CG-rich regions due to the complexities associated with determining the DNA’s secondary structure. Consequently, conventional PCR can only be employed to identify normal to pre-mutation alleles. To surmount this limitation, hybrid PCR methods have been developed which involving the incorporation of polymerase enzyme with solutions such as DMSO and betaine. Expand Long Template PCR is one such combined method, which can identify CGG repeats ranging from normal to full mutation and very large FMR1 alleles.

However, these methods are unable to detect homozygosity in women. To address this constraint, the hybrid PCR primer method was introduced, later evolving into the triplet repeat-primed PCR (TR-PCR) method. The TR-PCR method employs three types of primers, with two primers binding to sequences outside the CGG repeats and the other primer binding within the CGG regions. Simultaneous amplification occurs and the results are observed using the capillary electrophoresis technique. This method enables the detection of the number of repetitions in normal alleles up to full mutation.

PCR-based techniques, while capable of reproducing full mutation alleles, are unable to identify the epigenetic aspect of FXS disorder specifically the methylation state of the FMR1 gene. To address this limitation, methylation-specific PCR methods have been developed. One such approach involves inducing bisulfite modifications in CGG nucleotides and converting unmethylated cytosine to uracil. In this method, methylated cytosines remain resistant to modifications. By amplifying and sequencing the modified DNA, valuable insights into the methylation status can be obtained.

However, it is worth noting that this technique has its limitations, including the potential destruction of DNA structure as a result of bisulfite modifications. Nonetheless, despite these limitations, this method is routinely employed for FXS diagnosis and gene methylation analysis. Another alternative entail utilizing restriction enzymes that are sensitive to methylation, followed by replication and examination through capillary electrophoresis. In cases where individuals exhibit clinical symptoms of the disease but CGG measurement tests yield negative results, additional methods such as sequencing or multiplex ligation-dependent probe amplification (MLPA) can be employed for assistance. However, it is important to note that these methods are primarily applicable for FXS diagnosis in males. For females, it becomes imperative to determine the percentage of the inactive X chromosome, as it plays a pivotal role in the phenotype.

depending on the number of CGG repeats, some alleles that are not methylated may result in the production of FMRP, leading to less severe symptoms of the disease. Additionally, unmethylated alleles of FM have the potential to generate variations of FMR1 mRNA that can cause harm to carriers. Consequently, one of the methods utilized to diagnose Fragile X Syndrome (FXS) is the quantitative measurement of FMR1 transcripts and FMRP proteins. The technique of quantitative reverse transcription polymerase chain reaction (PCR) is extensively employed for this purpose.

In recent years, advancements in sensitive and high-resolution methods for the molecular investigation of FMR1 alleles have facilitated the evaluation of FMR1 methylation. Furthermore, there have been significant improvements in the quantification methods of FMR1 transcripts and FMRP proteins. These advancements have proven to be clinically advantageous in the diagnosis of Fragile X disorder and can aid in optimizing the categorization of patients for clinical trials.

Treatment of fragile X syndrome

Over the past decade, extensive research has been conducted on the development of treatments for Fragile X syndrome, some of which may be proposed as potential treatment modalities for this disorder in the future. However, thus far no definitive treatment method has been established for this disease. In general, treatment approaches for FXS can be classified into two distinct groups:

Reactivation of the damaged gene
Compensation for the deficiency of FMRP protein

Epigenetic Modulators

The strategy for the restoration of FMR1 gene activity focuses on reversing epigenetic changes through the presence of the gene’s coding sequence. Specifically, the strategy targets DNA methylation, which is potentially reversible. The initial compound tested on cells from FXS patients was 5-aza-deoxycytidine (also known as 5-azadC), a methyltransferase inhibitor. This compound was successful in restoring the transcription and translation of the FMR1 gene. Further studies have shown that the use of histone deacetylase inhibitors, such as TSA, butyrate and 4-phenylbutyrate enhances the effectiveness of azadC-5. Reactivation of the gene can be achieved not only through DNA demethylation, but also through modifications to the epigenetic code of histones H3 and H4. These changes resulted in the methylated FM allele becoming similar to the active UFM allele.

Initially, it was believed that azadC-5 specifically targeted the FMR1 gene, but it was discovered that it also affected other methylated genes and caused alterations. Additionally, due to its involvement in apoptosis induction azadC-5 cannot be easily utilized in the treatment of fragile X syndrome. Acetyl-L-carnitine (Nicetile), a natural compound that improves cellular metabolism, has been shown to inhibit cytogenetic expression of the fragile X locus in cultured lymphocytes from patients. Various clinical studies have revealed significant improvement in hyperactivity and adaptive behavior in patients. However, there was no change in FMR1 methylation status and gene expression did not increase. Another compound tested in the treatment of fragile X syndrome is valproic acid, which was previously recognized as a gene reactivator. However, it is only a weak activator of the FM gene.

GABAergic system

In patients with fragile X syndrome, the GABA receptor system experiences a downregulation phenomenon which can be attributed in part to the instability of mRNAs encoding GABA receptor subunits. Consequently, this downregulation leads to the disruption of GABAergic transmission in various regions of the brain such as the amygdala, striatum and cerebral cortex ultimately resulting in behavioral abnormalities associated with FXS disease. Certain pharmacological agents such as acamprosate, exert an influence on the GABAergic system by inhibiting NMDA-R and activating GABAA-R.

Targeting downstream targets of FMRP

Additional therapeutic approaches for fragile X syndrome encompass the regulation of protein production levels which are irregularly synthesized in the context of this disorder. One such protein is the glycogen synthase kinase 3β (GSK-3β) enzyme which is associated with mGluR1 signaling and is overexpressed in individuals affected by FXS. Notably, several studies have demonstrated that the utilization of lithium can lead to a reduction in the production of this enzyme.

In general, the continuous advancement of novel and targeted therapeutic modalities aimed at bolstering synaptic connections and enhancing patients’ well-being is an ongoing pursuit. Conversely, the utilization of induced pluripotent stem cells in animal models holds promise as a potential future treatment modality for fragile X syndrome, with the potential to ameliorate symptoms and address the underlying genetic disorder.