Fragile X Syndrome: A Review of Clinical and Molecular Diagnoses
Background
Fragile X Syndrome (FXS) is the most common inherited cause of intellectual disability (ID) and the most prevalent monogenic cause of autism spectrum disorder (ASD). It affects approximately 1 in 4000 males and 1 in 8000 females.
FXS is caused by a mutation in the FMR1 gene, located on the X chromosome at Xq27.3. This gene provides instructions for making the fragile X mental retardation protein (FMRP), which is essential for normal brain development.
In over 99% of cases, the mutation involves a CGG repeat expansion in the 5' untranslated region (UTR) of the FMR1 gene. Normal individuals have between 5 and 44 CGG repeats. In FXS, this repeat expands to over 200 repeats, leading to a full mutation (FM).
Less than 1% of cases are due to point mutations, deletions, or duplications within the FMR1 gene.
FXS, also known as Martin-Bell Syndrome, was first described in 1943 by Martin and Bell, who noted X-linked ID in a family.
In 1969, a fragile site on the X chromosome was linked to ID, providing a cytogenetic marker for the syndrome.
In 1991, the FMR1 gene was identified, and the association of Xq27.3 with X-linked ID was confirmed through molecular analysis.
Diagnosis is based on detecting an alteration of the FMR1 gene at Xq27.3, typically through PCR and Southern blot analysis to determine the CGG repeat number and methylation status.
More than 99% of individuals with FXS have a loss-of-function due to increased CGG repeats in the 5' UTR (typically >200 triplets), called a full mutation (FM). This expansion leads to transcriptional silencing of the FMR1 gene.
FM leads to hypermethylation of the FMR1 promoter, inhibiting transcription and consequently reducing or eliminating FMRP. This protein is crucial for synaptic plasticity and neural development.
Different FMR1 alterations (deletions, duplications, single-nucleotide variants) account for the remaining (<1%) of FXS cases. These can disrupt FMRP production or function through various mechanisms.
Normal CGG repeats in the FMR1 gene range from 5 to 44.
45-54 repeats: grey zone. Individuals with grey zone repeats are generally asymptomatic but may have a slightly increased risk of certain neurological or psychiatric conditions.
55-200 repeats: pre-mutation (PM), associated with premature ovarian failure (POI) in females and fragile X-associated tremor/ataxia syndrome (FXTAS) in males. PM carriers do not typically have intellectual disability but are at risk for these late-onset disorders.
This review focuses on the complete FXS phenotype with FM allele carriers, detailing the clinical presentation and molecular underpinnings of the syndrome.
FXS inheritance depends on the number of trinucleotide repeats. The expansion from PM to FM typically occurs during maternal (rarely paternal) X chromosome transmission. The risk of expansion increases with the number of CGG repeats in the pre-mutation allele.
Frequency of PM allele: approximately 1:850 in males and 1:257-300 in females. This high carrier frequency underscores the importance of genetic screening and counseling.
Affected men: ID, long face, large ears, macroorchidism (enlarged testicles), and behavioral problems such as hyperactivity, anxiety, and autism spectrum disorder.
Females heterozygous for the FM allele: 30% chance of normal IQ, 25% chance of ID (IQ < 70); can have learning deficits/emotional difficulties. The variable expression in females is due to X-chromosome inactivation.
The phenotype in females is strongly connected to the X inactivation (XCI) pattern. If the normal X chromosome is preferentially inactivated, the female will be more severely affected.
FXS: multi-systemic condition affecting any apparatus due to FMRP expression. FMRP is expressed in various tissues throughout the body, leading to a wide range of potential clinical manifestations.
The aim: to provide clinical and molecular knowledge about FXS for clinicians and lab workers, enhancing diagnostic accuracy and patient care.
General Clinical Features
Prenatal/neonatal diagnoses are usually not possible without a family history due to a lack of ultrasound and clinical findings. However, if a family history exists, prenatal testing can be performed via amniocentesis or chorionic villus sampling.
Height, weight, and head circumference are typically normal at birth. Affected infants usually do not show obvious signs/symptoms at birth
Macrocephaly develops in prepubertal age (head circumference >50th percentile). This becomes more apparent as the child grows, often noticeable by early childhood.
Facial characteristics become distinctive in early childhood (long narrow face, prominent ears). These features are subtle at first but become more pronounced over time.
Other facial features:
Prominent jaw
High-arched palate
Puffiness around eyes
Long palpebral fissures
Closely spaced eyes
Epicanthal folds
Strabismus
Flat nasal bridge
Broad nose
Broad philtrum
Facial hypotonia
Approximately 30% of young children with FXS do not have obvious dysmorphic features. This can make early diagnosis challenging, especially in the absence of a known family history.
The most important clinical abnormality is global developmental delay/ID. This is often the first sign that prompts clinical evaluation.
Psychomotor delay involves walking age (mean years) and age at first words (mean years). These milestones are typically delayed compared to neurotypical children.
Males and females: wide range of learning disabilities from normal/borderline IQ to severe ID. The severity of intellectual disability can vary widely.
IQ of males with FM varies, mean . 68% have IQ < 50, 18% have IQ > 70. This distribution highlights the range of cognitive abilities among affected males.
IQ directly correlates with FMRP production. The more FMRP that is produced (even if at reduced levels), the higher the IQ tends to be.
Females with FM: wider range of phenotypic characteristics depending on XCI pattern; 70% have some cognitive impairment. Due to X-inactivation, some females may have near-normal cognitive function.
Neurological Features
Epilepsy:
Prevalence in FXS children: 10-20% in boys, 5-10% in girls. Seizures are a common neurological comorbidity.
Complex partial seizures are most frequent (89.3%). These seizures involve impaired awareness and complex motor or sensory phenomena.
Generalized tonic-clonic seizures (46.4%). These seizures involve loss of consciousness and violent muscle contractions.
Simple partial seizures (25%). These seizures involve preserved awareness with motor, sensory, or psychological symptoms.
Febrile convulsions (7.1%). These are seizures triggered by fever, more common in young children.
Onset: usually between 2 and 10 years; typically disappears with growth (25% continue into adulthood). Seizures often resolve or become less frequent as the child matures.
Low frequency of recurrence; can manifest following infections or environmental factors. Seizure triggers can include illness, stress, or sensory overload.
Good response to therapy. Most individuals with FXS-related epilepsy respond well to antiepileptic medications.
Most patients control seizures with antiepileptic drugs (AEDs); 7% need multiple drugs, 10% need no therapy. This indicates that a significant proportion of individuals may not require medication.
EEG abnormalities (74%) are more prevalent, independent of epilepsy. Even in the absence of clinical seizures, EEG can reveal underlying neurological dysfunction.
Slowing of the posterior dominant rhythm (47%). This is a common finding, reflecting altered brain activity.
Focal spikes from various anatomic regions (42%). These spikes indicate areas of cortical irritability.
Normalization of EEG background after age 8 (35%). In some cases, EEG abnormalities may improve with age.
MRI is usually normal. Structural brain abnormalities are not always apparent on standard MRI.
When anomalies are found: diffuse atrophy, cortical thickness, increased cortical volume and complexity. These findings suggest altered brain development and connectivity.
Consistent with decreased pruning, increased spine density/length, and immature spine. These microscopic changes affect synaptic function and plasticity.
Other findings: atrophy of cerebellar vermis, thinning of corpus callosum, hippocampal anomalies, enlarged fourth ventricle, lacunar infarction of basal ganglia, mesial temporal sclerosis. These less common findings can contribute to the neurological phenotype.
Increased fractional anisotropy in specific brain regions, correlating with cognitive performance. This suggests altered white matter organization.
Neuropsychiatric Features
ID is rarely presented alone in FXS. Individuals typically have a range of behavioral and psychiatric issues.
Psychomotor delay is an early sign. Delays in motor and cognitive development are often the first indicators.
Developmental profile deviates by 6 months of age, involving all domains. Early intervention is crucial to address these developmental delays.
Average functional level improves until age 25, then stabilizes until 50, then worsens. This reflects the natural course of development and aging in individuals with FXS.
High risk of developing neuropsychiatric disorders. Psychiatric comorbidities are common and significantly impact quality of life.
Association between autism and FXS was noted in the early 1980s. This association is well-established in the literature.
FXS/autism and FXS/ADHD are the most studied associations. These are the most common and extensively researched psychiatric comorbidities.
Autism Spectrum Disorder (ASD) comorbidity: 30-50% males, 25% females. The prevalence of ASD is significantly higher in males with FXS.
Age-related improvement in some ASD symptoms in FXS men (diagnosed in approximately 49% of children but 41% of adolescents/adults). Some autistic traits may lessen with age.
ASD-related impairment is less severe in FXS individuals than in those with non-syndromic ASD. The overall impact of autism may be less pronounced in FXS.
Changes in DSM criteria (DSM-IV TR to DSM-5 in 2013) are modifying the rate of incidence. Diagnostic criteria changes can affect prevalence rates.
All children with ASD, especially boys, should be tested for FMR1. Screening for FXS is recommended in all cases of unexplained ASD.
ADHD is a common comorbidity. Attention deficits and hyperactivity are frequently observed.
Prevalence of ADHD spectrum symptoms: 54-59%. A significant proportion of individuals with FXS exhibit ADHD symptoms.
Complete diagnostic criteria fulfilled by 12-23% of FXS subjects. Formal ADHD diagnosis is less common than the presence of ADHD symptoms.
Preschool boys do not differ from typically developing controls in ADHD symptoms, peak at school age (5-6 years). ADHD symptoms tend to become more prominent during school years.
Hyperactivity incidence: 50-66%. Hyperactive behaviors are common.
Attention problems prevalence: 74-84%. Difficulties with attention are highly prevalent.
Anxiety disorder prevalence: 58-86%. Anxiety is a significant issue for many individuals with FXS.
Increased with age. Anxiety may worsen with age and increased awareness of social challenges.
Depression prevalence: 8-12% in adolescents/adults. Depression can manifest as withdrawal or aggression.
May manifest as withdrawal or increased aggression. Atypical presentations of depression are common.
Other common neuropsychiatric conditions:
Pervasive developmental disorder.
Stereotypies (mostly hand/finger mannerisms). Repetitive motor behaviors are frequently observed.
Sleep problems. Sleep disturbances are common and can exacerbate other behavioral issues.
Specific or social phobias. Irrational fears can significantly impair daily functioning.
Selective mutism. Difficulty speaking in certain social situations is sometimes seen.
Restricted interests. Narrow focus on specific topics or activities is typical.
Compulsive and ritualistic/sameness behaviours. These behaviors can interfere with daily routines.
Self-injurious behaviour. This can occur in more severe cases.
Aggressiveness (90% of individuals with FXS). Aggression can be a challenging behavior to manage.
Sleep disorders affect approximately 30% of FXS children. These disorders can include insomnia, sleep apnea, and night terrors.
Using DSM-IV-TR criteria, 73% of FXS patients can be diagnosed with at least one axis I psychiatric disorder. This underscores the high rate of psychiatric comorbidity.
The assessment of psychiatric symptoms is often complicated by limitations in self-reporting and insight, atypical manifestation of symptoms, and lack of validated assessment tools. Clinicians need to be aware of these challenges when evaluating psychiatric symptoms.
Diagnostic overshadowing occurs when psychiatric symptoms are attributed only to the disability itself. This can lead to underdiagnosis and undertreatment of psychiatric disorders.
A Real Syndrome: Multi-Systemic Involvement
FXS shows an association of various medical problems that can worsen the phenotype and complicate the clinical management. A holistic approach to care is essential.
Shares some features with the connective tissue disorder (CTD) spectrum, but a specific abnormality has not yet been shown. Joint hypermobility and soft skin are common.
Skin can be soft. This can contribute to skin sensitivity.
Joint hypermobility is present in about half of the patients, affecting predominantly the small joints. This can lead to joint pain and instability.
Skeletal signs may include a high-arched palate, scoliosis, pectus excavatum, and flat feet. These skeletal abnormalities are relatively common.
Cardiac defects:
Aortic root dilatation (approximately 25% of the patients). Regular monitoring is needed to prevent aortic dissection.
Mitral valve prolapse (3-50%). This condition can cause heart murmur and, in some cases, require intervention.
Hyperarousal and reduced parasympathetic vagal tone. - These autonomic nervous system imbalances can contribute to anxiety and cardiac issues.
In adult age (>40 years), FXS patients tend to develop common cardiovascular problems such as hypertension (24.2%) and heart rhythm disorders (24.2%). Regular cardiovascular screening is important.
Gastrointestinal problems:
Gastro-oesophageal reflux, constipation, and loose bowel movements. These issues can significantly affect comfort and nutrition.
A prevalence of gastrointestinal problems of 30.6% was reported in FXS males and females aged 40–71 years. GI issues persist into adulthood.
Genitourinary system:
Pubertal macroorchidism in males (80-95% of adults). Enlarged testicles are a characteristic feature.
Mean testicular volume: approximately 50 mL (normal mean: <25 mL). This is significantly larger than normal.
Most FXS men do not have any real fertility complaints, even if 1% have been reported to reproduce. Despite enlarged testicles, fertility is usually not significantly affected.
Ocular anomalies:
Higher prevalence of strabismus (8-40%) and refractive errors (17-59%). Vision problems are common.
Nystagmus (5-13%). Involuntary eye movements can affect vision.
Other features include palpebral ptosis and convergence insufficiency. These can contribute to visual discomfort and fatigue.
FXS children tend to have recurrent otitis media, which may lead to conductive hearing loss. Early management of ear infections is important to prevent hearing issues.
Metabolic problems:
Obesity and overweight (53-61%). Weight management is important for overall health.
In males, the serum levels of HDL are shifted to lower numbers across all age ranges, but conversely, their triacylglycerol levels are higher than those of the general population. This lipid profile increases cardiovascular risk.
Differential Diagnosis
Includes syndromic forms of ID and non-syndromic psychomotor delays/ID. It is important to distinguish FXS from other conditions with similar features.
Conditions with shared phenotypic features:
Sotos Syndrome: ID, macrocephaly, behavioural problems, and epilepsy. Sotos syndrome involves overgrowth in childhood.
Prader-Willi Syndrome: developmental delay, facial appearance elements, sucking problems in neonatal age, obesity, and genital anomalies. Prader-Willi syndrome is characterized by hyperphagia and hypotonia.
Klinefelter Syndrome: ID (20%) and genital anomalies. Klinefelter syndrome affects males and involves an extra X chromosome.
FRAXE: ID (generally milder than FXS), language impairment, hyperactivity, and autistic behaviour. FRAXE is another form of X-linked intellectual disability.
Angelman and Rett Syndromes may also be considered. These syndromes involve distinct genetic and clinical features.
Array-CGH can be performed to exclude cytogenetic rearrangements. This technique can identify deletions or duplications of chromosomal regions.
When genetic testing is not helpful, isolated ID, autism, or ADHD must be considered. In the absence of a clear genetic diagnosis, these conditions should be evaluated and managed.
The FMR1 Gene and FMRP: From Triplet Expansion to Pathology
FXS is associated with a fragile site on Xq27, named FRAXA. This cytogenetic marker led to the discovery of the FMR1 gene.
In 1991, an association between FXS and alterations of the FMR1 gene was identified at the FRAXA locus. This discovery revolutionized the diagnosis and understanding of FXS.
FMRP is involved in the regulation of post-transcriptional RNA metabolism, playing an important role in synaptic plasticity, dendrite and axon development, and underlying learning and memory. The absence of FMRP disrupts these critical processes.
The absence of FMRP derives, in most cases, from a dynamic mutation consisting of variable expansion of a trinucleotide (CGG) repeat in the 5′ UTR of the FMR1 gene. This expansion leads to transcriptional silencing.
The size of the CGG repeat in normal individuals ranges between 5 and 44. These individuals produce normal levels of FMRP.
Alleles with 45–54 repeats are defined as intermediate, borderline or “grey-zone” (GZ). These individuals are generally asymptomatic but may have subtle neurological or psychiatric issues.
Carriers of GZ alleles do not show an FXS phenotype but can present with peripheral neuropathy, ataxia, anxiety and/or depression, and clinical symptoms similar to those of Parkinson patients. These symptoms are less severe than those seen in PM or FM carriers.
PM carriers have a number of repeats that ranges from 55 to 200. PM carriers are at risk for late-onset disorders.
PM disorders were first identified in 1991 with the discovery of an increased incidence of early menopause (prior to the age of 40 years) in female carriers. This led to further research on the effects of PM expansions.
Twenty percent of PM females manifest an FMR1-related POI. POI can have significant implications for fertility and overall health.
FXTAS occurs in PM males (rarely in females) and is characterized by late-onset, progressive cerebellar ataxia and intention tremor. FXTAS is a debilitating neurological condition.
The necessary findings to confirm an FXTAS diagnosis are an FMR1 PM associated with an MRI showing white matter lesions in the middle cerebellar peduncles and/or brain stem and intention tremor or gait ataxia. MRI and clinical findings are essential for diagnosis.
In FXS patients, the CGG trait is expanded (FM), with a dimension greater than 200 repeats. This expansion leads to complete or near-complete loss of FMRP.
This expansion results in transcription silencing and the consequent absence of FMRP, due to hypermethylation of the CpG islands adjacent to the expanded trinucleotide repeats and heterochromatin conformation of the FMR1 promoter region. Epigenetic modifications prevent FMR1 gene expression.
The risk of a PM allele becoming an FM allele is correlated with the number of CGG trinucleotide repeats, with nearly all alleles with >=100 repeats expanding to FM in the next generation when transmitted by the mother. The higher the repeat number, the greater the risk of expansion.
Beyond CGG repeat size, one of the major factors influencing FMR1 stability is the presence of AGG triplets interspersed within the FMR1 repeated region. AGG interruptions reduce the risk of expansion.
Maternal alleles with no AGGs have the greatest risk for FM expansion. These alleles are highly unstable.
A decrease in CGG repeat number through generations, although rare, is also possible. This is an uncommon phenomenon.
Mosaicism is a source of phenotypic variability, and it can be observed in both sexes, with a higher incidence in males. Different cells can have different CGG repeat numbers.
In addition to the FMR1 promoter expansion, a small number of FXS cases (<1%) are caused by mutation in the coding region or deletion of the FMR1 gene. These alterations directly disrupt FMRP function or production.
Another important issue to consider, when approaching a diagnosis in females, is X chromosome inactivation (XCI), consisting of the silencing of one of the two X chromosomes in mammalian females. XCI patterns can significantly affect the phenotype in females.
Diagnostic Procedures
Initially, the diagnosis of FXS was based on the cytogenetic evaluation of the presence of FRAXA in peripheral blood lymphocytes (PBLs). This method was limited by its low sensitivity and specificity.
Cytogenetic analyses were replaced by Southern blot analysis of DNA from peripheral blood after digestion with specific restriction endonucleases and finally by PCR. These molecular techniques allowed for more accurate diagnosis.
Southern blot analysis can detect all FMR1 alleles, including normal, PM, and FM, and can determine the methylation status of the FMR1 promoter region; however, it is time consuming, relatively expensive, and similarly to the previously used approaches, difficult to interpret. Southern blot is still used for confirming results and determining methylation status.
Standard PCR plus Southern blot analysis has been considered the gold standard for FMR1 molecular diagnosis for a long time, even if it provides a low-resolution estimation of the repeat number. This combination provided both repeat sizing and methylation analysis.
Triplet primed PCR (TP- PCR) was designed: it is a procedure in which the forward PCR primer is located upstream the CGG region and the other overlaps the CGG repeat and the adjacent unique sequence; after PCR cycles, the CGG repeat number can be determined by fragment sizing of PCR amplicons using capillary electrophoresis. TP-PCR allows for rapid and accurate sizing of CGG repeats.
As the second-level analysis in the diagnostic flow- chart, CGG methylation testing can be performed to evaluate FMRP silencing. Methylation analysis confirms transcriptional silencing of the FMR1 gene.
FMR1 sequence analysis and MLPA must therefore be offered to patients with a clinical phenotype highly suggestive of FXS but with a normal range of CGG repeats. These tests can detect rare mutations or deletions in the FMR1 gene.
Prenatal FMR1 Testing
FXS molecular tests are usually performed postnatally on PBLs in the presence of the appropriate clinical criteria described in the clinical section. However, prenatal testing is available for at-risk pregnancies.
Moreover, it is also possible to perform a prenatal test of FXS using LR- PCR-based protocols on DNA from either chorionic villi or amniocytes. These procedures carry a small risk of miscarriage.
FMR1 prenatal testing should be offered to couples with a personal or familial history of the following:
FXS- or FX-related disorders
Unexplained ID or developmental delay
Isolated cognitive impairment
Autism
Idiopathic familiar POI or elevated FSH at age <40 years
Isolated cerebellar ataxia with tremor
Considering all of these issues, for couples who re- quest FXS screening, the ideal test should be proposed in the preconception period: this could be advantageous because the couple will be allowed to make conscious reproductive decisions. Preconception counseling and testing allow for informed reproductive choices.
Newborn screening for FXS has been proposed, even if its application remains controversial. The benefits and ethical considerations of newborn screening are debated.
Patient Management: Therapeutic Strategies and Social Issues
FXS emerges as a complex disease with a primary neuro- psychiatric involvement but potentially affects more than one apparatus, therefore needing a large-scale intervention able to address all the physical, psychological and social implications of the disease. Comprehensive, multidisciplinary care is essential.
Treatment plans should be individualized based on the symptoms and age-related comorbidities of each individual. There is no one-size-fits-all approach.
Speech and language therapy must be recommended to children, especially in that with early diagnosis. Early intervention can improve communication skills.
Behavioural therapy is another useful tool that helps normalize some of the symptoms, and it has been reported to be an effective approach for aggression in 71% of patients. Behavioral interventions can address a variety of symptoms.
Physical therapy is needed in some cases, and occupational therapy must be offered to adults. These therapies can improve motor skills and daily functioning.
Pharmacologic therapy may be recommended to improve behaviour problems (such as aggression, anxiety, hyperactivity, problems with impulse control, and poor attention span) and also to treat more severe disorders, such as ADHD or depression. Medications can help manage specific symptoms.
Lithium has been proposed as possible treatment, and in 2008, a pilot add-on trial has been conducted to evaluate the safety and efficacy of this drug in FXS pa- tients. Research continues to explore new treatment options.
New targeted treatments for FXS (mGluR5 antagonists, GABA A and B agonists, minocycline) are now being studied. These treatments target specific molecular pathways.
Going beyond treatment, an important issue to con- sider in the management of FXS patients is the psycho- logical health of their parents and caregivers; both have to take care of these complicated patients in different ways, and both have been proven to suffer psychological consequences. Support for families and caregivers is crucial.