Fragile X syndrome: A Review with special emphasis on molecular genetics

Fragile X syndrome is a genetic disorder, which is due to a mutation in the X-linked FMR1 gene that causes a range of developmental problems including learning disabilities and cognitive impairment or intellectual disability. Males with fragile X syndrome almost always exhibit mental retardation, usually in the moderate range, and often have characteristic physical features and behaviour. Since the mutation is X-linked, males are more severely affected than females.

Prevalence:

In general population Fragile X syndrome occurs in approximately 1 in 4,000 (1:4000) males and 1 in 8,000 (1:8000) females.

Signs and symptoms of Fragile X syndrome:

1.       Physical features
The physical features include a long and narrow face (elongated face), large and protruding ears, flexible fingers, flat feet, macroorchidism (large testicles) and low muscle tone (hypotonia). During early childhood, recurrent otitis media (middle ear infection) and sinusitis is common. Speech may be cluttered or nervous. Behavioral characteristics may include stereotypic movements (e.g., hand-flapping) and atypical social development, particularly shyness, limited eye contact, memory problems, and difficulty with face encoding. About a third of those affected have features of autism such as problems with social interactions and delayed speech. Hyperactivity is common and seizures occur in about 10%. Males are usually more affected than females.
2.       Intellectual development:
Studies show that Individuals with Fragile X syndrome have low IQ level, with an average IQ of 40 in males who have complete silencing of the FMR1 gene but in case of females, who are less vulnerable to FXS, generally have an IQ which is normal or borderline with learning difficulties.
The main difficulties in individuals with FXS are with working and short-term memory, visual-spatial relationships, visual memory, verbal abilities being relatively spared, executive function and low mathematics ability.
3.       Autism:
Most of the time Fragile X syndrome co-occurs with autism in many cases and FXS is a suspected genetic cause of the autism in these cases. Prevalence of concurrent autism spectrum disorder (ASD) in individual with FXS has been estimated to be between 15 and 60%.
In different studies, genetic mouse models of FXS have also been shown to have autistic-like behaviours.
4.       Social interaction:
Social anxiety is one of the most common features associated with FXS. FXS is characterized by social anxiety, including poor eye contact, gaze aversion, prolonged time to commence social interaction, and challenges forming peer relationships. Social anxiety in individuals with FXS is related to challenges with face encoding i.e. the ability to recognize a face that one has seen before. Females with FXS frequently display shyness, social anxiety and social avoidance or withdrawal.
5.       Mental health:
The most common psychiatric diagnosis in those with FXS is the “Attention deficit hyperactivity disorder” (ADHD). Children with fragile X have very short attention spans, are hyperactive, and show hypersensitivity to visual, auditory, tactile, and olfactory stimuli.
The other common communicative and behavioural characteristic in FXS is Perseveration. Children with FXS may repeat a certain ordinary activity over and over. In speech, repetition of the same phrase, also talking about the same subject continually, cluttered speech and self-talk are commonly seen. Self-talk generally includes chatting with oneself using different tones and pitches.
Fragile X-associated tremor/ataxia syndrome (FXTAS) is characterized by problems with movement and thinking ability (cognition). FXTAS is a late-onset disorder, usually occurring after age 50, and its signs and symptoms worsen with age. This condition affects males more frequently and severely than females. Affected individuals have areas of damage in the part of the brain that controls movement (the cerebellum) and in a type of brain tissue known as white matter, which can be seen with magnetic resonance imaging (MRI). The characteristic features of FXTAS are intention tremor, which is trembling or shaking of a limb when trying to perform a voluntary movement such as reaching for an object, and problems with coordination and balance (ataxia).
6.       Neurology:
Several studies reported that individuals with FXS are at a higher risk of developing seizures, with rates between 10% and 40%. In larger study populations the frequency varies between 13% and 18%, recent studies also found that 14% of males and 6% of females experienced seizures.
7.       Working memory
Males with FXS begin developing progressively more severe problems at the age of 40 onwards in performing tasks that require the central executive of working memory. The working memory implicates the temporary storage of information 'in mind', while processing the same or other information. Phonological memory (or verbal working memory) deteriorates with age in males, while visual-spatial memory is not found to be directly related to age.
Studies shows that the CGG length is significantly correlated with central executive and the visual–spatial memory. However, in a premutation individual, CGG length is only significantly correlated with the central executive, not with either phonological memory or visual–spatial memory.
8.       Fertility
In female who are carriers for the fragile X premutation about 20% of them are affected by fragile X-related primary ovarian insufficiency (FXPOI), which could defined as menopause before the age of 40.
Molecular basis of Fragile X syndrome
Fragile X mental retardation 1 (FMR 1) gene gives rise to a family of disorders when its non-coding CGG-repeat element is expanded to either the premutation range (55–200 CGG repeats) or the full mutation range (>200 CGG repeats). Alleles that have 45–54 CGG repeats are labeled as the “gray zone” because instability in transmission to the next generation is common. The molecular basis of the syndrome is usually an expansion of a repetitive CGG triplet sequence located in the 5’-untranslated region (5’-UTR) of FMR1 gene. Rarely, FXS results from other molecular alterations (such as point mutations or deletions) within FMR1 gene. The CGG repeat element is polymorphic, varying from 6 to 44 repeats in the normal range, from 45 to 54 repeats in the gray zone, and from 55 to 200 repeats in the premutation range. For alleles below the gray zone, CGG repeat is generally stable in parent-to-offspring transmissions. However, CGG elements in the premutation range become increasingly unstable with increasing repeat number, and alleles exceeding 59 CGG repeats can expand to a full mutation in a single generation, almost exclusively by transmission from mother to son. The expansion of the number of repeats above a threshold of approximately 200 repeats, results in hypermethylation of FMR 1 promoter region and a lack of gene expression which fail to express FMRP protein, this allele is termed as full mutation. The premutation is relatively common in the general population, present in approximately 1 in 130 to 250 women and 1 in 250 to 810 males. The full mutation is less common at approximately 1 per 2,500 to 1 per 4,000. Mothers who bring their children into the clinic are usually without apparent clinical involvement cognitively, although anxiety and depression are common. Variation in FMR1 CGG repeat size is a useful biomarker of various types of risk that could affect parents, as it defines differences between “healthy” and “affected” and between full mutation and premutation carriers.
Pattern of Inheritance
Fragile X syndrome is inherited in an X-linked dominant pattern. A condition is considered X-linked if the mutated gene that causes the disorder is located on the X chromosome, one of the two sex chromosomes. The Y chromosome is the other sex chromosome. The inheritance is dominant if one copy of the altered gene in each cell is sufficient to cause the condition. X-linked dominant means that in females who have two X chromosomes, a mutation in one of the two copies of a gene in each cell is sufficient to cause the disorder. In males who have only one X chromosome, a mutation in the only copy of a gene in each cell causes the disorder. In most cases, males experience more severe symptoms of the disorder than females.
 In women, the FMR1 gene premutation on the X chromosome can expand to more than 200 CGG repeats in cells that develop into eggs. This means that women with the premutation have an increased risk of having a child with fragile X syndrome. By contrast, the premutation in men does not expand to more than 200 repeats as it is passed to the next generation. Men pass the premutation only to their daughters. Their sons receive a Y chromosome, which does not include the FMR1 gene.
Pathophysiology of Fragile X syndrome
Synaptic plasticity:
The Fragile X mental retardation protein (FMRP) has a diverse array of functions throughout different areas of the neuron; however these functions have not been fully characterized. FMRP has been suggested to play roles in nucleocytoplasmic shuttling of mRNA, dendritic mRNA localization, and synaptic protein synthesis. FMRP is found throughout the human body. It is reported that brain and testes have the highest concentrations of FMRP. It appears to be primarily responsible for selectively binding to around 4% of mRNA in mammalian brains and transporting it out of the cell nucleus and to the synapses of neurons. Most of these mRNA targets have been found to be located in the dendrites of neurons, and brain tissue from humans with FXS and mouse models shows abnormal dendritic spines, which are required to increase contact with other neurons. The subsequent abnormalities in the formation and function of synapses and development of neural circuits result in impaired neuroplasticity, an integral part of memory and learning. Connectome changes have long been suspected to be involved in the sensory pathophysiology and most recently a range of circuit alterations have been shown, involving structurally increased local connectivity and functionally decreased long-range connectivity.
 Group 1 metabotropic glutamate receptor (mGluR) pathway:
Group 1 metabotropic glutamate receptor (mGluR) signaling has been implicated in playing an important role in FMRP-dependent synaptic plasticity. Post-synaptic mGluR stimulation results in the up-regulation of protein synthesis through a second messenger system. A role for mGluR in synaptic plasticity is further evidenced by the observation of dendritic spine elongation following mGluR stimulation. Furthermore, mGluR activation results in the synthesis of FMRP near synapses. The produced FMRP associates with polyribosomal complexes after mGluR stimulation, proposing the involvement of fragile X mental retardation protein in the process of translation. This further advocates a role for FMRP in synaptic protein synthesis and the growth of synaptic connections. The loss of FMRP results in an abnormal dendritic spine phenotype.
Effect of FMRP on dopamine pathway:
FMRP also appears to affect dopamine pathways in the prefrontal cortex which is believed to result in the attention deficit, hyperactivity and impulse control problems associated with FXS.
Effect of FMRP on GABA pathway:
The downregulation of GABA pathways, which serve an inhibitory function and are involved in learning and memory, may be a factor in the anxiety symptoms which are commonly seen in FXS.
Diagnosis of Fragile X syndrome
FXS can be diagnosed by testing a person’s DNA from blood. Diagnosis of FMR1 mutation while the fetus is in the uterus could be performed with chorionic villus sampling or amniocentesis. The following techniques are used for the diagnosis of Fragile X syndrome:
Culturing cells in a folate deficient medium:
 This is was of the oldest technique to diagnose FXS. This technique is very unreliable and not used now a days. Through this technique FXS and carrier status could be determined by culturing cells in a folate deficient medium and then assessing for "fragile sites" on the long arm of the X chromosome.
PCR and Southern blotting:
Fragile X genotyping can be done by direct PCR amplification of the CGG trinucleotide repeats region or by southern analysis. In most cases both methods are used to complement the results. Full mutations usually cannot be identified by PCR and southern analysis is the preferred method to distinguish full mutations.
Single-molecule real-time sequencing (SMRT)
Single-molecule real-time sequencing (SMRT) is a single molecule DNA sequencing method. Because the above method only tests for expansion of the CGG repeat, individuals with FXS due to missense mutations or deletions involving FMR1 will not be diagnosed using this test and should therefore undergo sequencing of the FMR1 gene if there is clinical suspicion of FXS.
Commercial Kit for the diagnosis of Fragile X syndrome
1.       CpG WIZ® Fragile X Amplification Kit (Catalogue No. S7807; Merckmillipore)
2.       AmplideX® mPCR FMR1 (Asuragen Genetics)
3.       Fragile X GScanTM V2 6-FAM Kit; 1 Kit (Catalogue No. 40-2004-15FM; Genelink)
Management of Fragile X syndrome
Therapy
There is no cure yet for the FXS. Management of FXS may include: speech therapy, behavioral therapy, sensory integration occupational therapy, special education, or individualised educational plans, and, when necessary, treatment of physical abnormalities.
 Medication
Present trends in treating the FXS include medications for symptom-based treatments that aim to minimize the secondary characteristics associated with the disorder.
For co-morbid disorders with FXS, antidepressants such as selective serotonin reuptake inhibitors (SSRIs) are utilized to treat the underlying anxiety, obsessive-compulsive behaviours, and mood disorders. Following antidepressants, antipsychotics such as risperidone and quetiapine are used to treat high rates of self-injurious, aggressive and aberrant behaviours. Anticonvulsants are another set of pharmacological treatments used to control seizures as well as mood swings. Drugs targeting the mGluR5 (metabotropic glutamate receptors) that are linked with synaptic plasticity are especially beneficial for targeted symptoms of FXS. Lithium is also currently being used in clinical trials with humans, showing significant improvements in behavioural functioning, adaptive behaviour, and verbal memory. Few studies also suggested using folic acid.
ADHD, which affects the majority of boys and 30% of girls with FXS, is frequently treated using stimulants. However, the use of stimulants in the fragile X population is associated with a greater frequency of adverse events including increased anxiety, irritability and mood lability. Anxiety, as well as mood and obsessive-compulsive symptoms, may be treated using SSRIs, although these can also aggravate hyperactivity and cause disinhibited behaviour.
Current Clinical trials and Research
Evidence from mouse models shows that mGluR5 antagonists (blockers) can rescue dendritic spine abnormalities and seizures, as well as cognitive and behavioural problems, and may show promise in the treatment of FXS. Two new drugs, AFQ-056 (mavoglurant) and dipraglurant, as well as the repurposed drug fenobam are currently undergoing human trials for the treatment of FXS. There is also early evidence for the efficacy of arbaclofen, a GABAB agonist, in improving social withdrawal in individuals with FXS and ASD. In addition, there is evidence from mouse models that minocycline, an antibiotic used for the treatment of acne, rescues abnormalities of the dendrites. An open trial in humans has shown promising results.
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