FRAGILE-X SYNDROME
Manga Sabaratnam

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Fragile-X syndrome is an X-linked, semi-dominant disorder with reduced penetrance. In addition to being associated with characteristic physical and behavioural features, it causes intellectual disabilities ranging from mild to severe. It is the most common cause of inherited intellectual disability and is second only to Down's syndrome as the most common genetic cause of intellectual disability. There are about 100-200 affected births in the UK each year. The population prevalence is estimated at 1 in 4000 males and 1 in 8000 females. It occurs in all races and ethnic groups. It accounts for approximately 10% of all males with severe intellectual disabilities and 10% of mild intellectual disabilities. There is a wide spectrum of clinical features, but life expectancy is not greatly reduced.

Fragile-X syndrome was first documented by Martin and Bell in 1943. They described a pedigree with 11 affected males and four mildly affected females in three generations. In 1972, this pedigree was re-examined, along with a large number of others, and it was proposed that the large number of males affected pointed to an X-linked condition. The "break" just above the tip of the X chromosome's long arm (the fragile site) was first demonstrated microscopically in 1969 by Lubs, but was not confirmed until 1977, with the discovery that a folate-deficient culture medium was required to show the fragile site. The fragile site is at region Xq27.3 on the long arm of the X chromosome (Figure 1).

In 1991 the long-awaited FMR-1 (fragile-X mental retardation) gene was isolated using a molecular genetic positional cloning strategy. The mutational basis of the syndrome (termed FRAXA) was recognized to be due to the expansion of a trinucleotide repeat (CGG)n present in the 5' untranslated region of the identified gene. The lack of expression of the FMR-1 gene results in non-production of the protein (fragile-X mental retardation protein, or FMRP), resulting in fragile-X syndrome. FMR-1 is the first cloned gene to be linked to human intelligence.

The FMR-1 gene and its mutations
Ninety-five per cent of cases of fragile-X syndrome are due to expansion in the CGG sequence. Normal individuals carry between 5 and 54 copies of CGG repeat. In normal carriers, the number of CGG repeats (the premutation) is between 55 and 200. In individuals clinically manifesting the syndrome, the CGG repeat (the full mutation) increases to 200-2000 or more. Such a large mutation is usually accompanied by hypermethylation of the DNA sequence, whereby methyl groups attach to the CGG triplets. This renders the FMR-1 gene transcriptionally inactive.

It is also possible for the CGG expansions to vary from cell to cell, resulting in somatic heterogeneity in allele size. Between 12% and 40% of affected males are so-called 'mosaics' - i.e. they exhibit both a premutation and a full mutation in the blood. In a 'size mosaicism', an individual with a full mutation also has pre­mutation cell lines; in a 'methylation mosaicism', a proportion of those with a full mutation in every cell have cell lines in which the FMR-1 gene is either partially or completely unmethylated; or the size of the full mutation can vary between different cell lines within an individual.

Fragile-X mental retardation protein (FMRP)
Under normal circumstances, the FMR-1 gene encodes FMRP. The absence of FMRP is likely to be responsible for the development of the fragile-X phenotype (see below). The exact role of FMRP is unknown, but it is thought to act to regulate protein synthesis by facilitating nucles-cytoplasmic transmission of genetic material, and messenger RNA - rhibosomal binding. FMRP usually helps the connection between neurons that underlie learning and memory; absence of FMRP seems to delay the development of such neurons. In normal individuals, FMRP is ubiquitously expressed, but at higher levels in the brain and the testes. The major characteristics in affected individuals with fragile-X syndrome relate to the functioning of the brain, and macro-orchidism (testicular enlargement). People with the premutation make FMRP; those with the full mutation do not. Females with the full mutation on one X chromosome and normal FMR-1 on the other make a reduced amount of FMRP.

FIGURE 1: The X chromosome in fragile-X syndrome. The arrow indicates the fragile site.

Genetic testing
As the fragility at Xq27.3 is visible under the microscope in only 4-50% of cells, cytogenetic testing has been superseded by more accurate DNA tests. These are less expensive, less time-consuming and can accurately detect both full mutation and premutation and provide details about allele size and methylation in affected individuals as well as normal-transmitting males and carrier females. In the UK, the two tests to identify the FMR-1 gene mutation are Southern blot of EcoRI-digested DNA and polymerase chain reaction (PCR).

• PCR is the routine screening test used on fragile-X samples. It is particularly effective for small increases (premutation) but is not very sensitive in detecting full mutations.
• If PCR fails, Southern blotting is performed. This can detect full mutations and methylation status of the regulatory site and the presence of mosaicism. It is more labour-intensive than PCR and requires larger amounts of genomic DNA. Southern blot analysis detects alleles in all size ranges, but precise sizing is not possible. The technique looks for amplification of the length of the FMR-1 gene.

More recently, an FMRP antibody test has been developed to measure expression in lymphocytes. This can detect the full mutation in males. It is not useful in mosaic males or females as some FMRP is still formed. Although a faster and less expensive way of screening males with undiagnosed causes of intellectual disability, it is not yet widely available in the UK.

Other fragile sites
Of the other nearby fragile sites identified on the X chromosome, namely FRAX-D, FRAX-E and FRAX-F, only the FRAX-E mutation is associated with intellectual disability (Figure 2). Distinguishing these sites from FRAX-A on standard chromosome cultures has become easier with improving techniques, especially fluorescent in situ hybridization (FISH).

Screening
The discovery of the FMR-1 gene means that, theoretically, DNA-based screening for the premutation could forewarn all potentially affected families. Population-based screening is neither feasible nor ethical mainly because of the current inability to distinguish full-mutation female fetuses with mental impairment from fetuses whose intelligence is not affected. Therefore, screening would be targeted at individuals who are at a higher risk. The various proposed strategies include preconceptual testing and routine prenatal screening of all carrier pregnancies. Other strategies could include systematic testing in affected families ('cascade' screening or extended family follow-up), case finding in paediatric or adult practice, or a combination of these.

Barriers to implementing even limited paediatric screening and cascade screening in affected families include inadequate resources, difficulties in counselling those with intermediate-range alleles, and the need to achieve uniformly high standards in existing screening programmes as a prerequisite for any population-based programme.

FIGURE 2:

Inheritance and genetic counselling
The identification of the FMR-1 gene has led to an increased understanding of the unusual and previously unexplained features of inheritance. Fragile-X syndrome is an X-linked, dominantly inherited disorder, with reduced penetrance, but its pattern of inheritance is atypical.

Firstly, both females and males can be affected, although it is less common and often less severe in females. In addition, both males and females can be unaffected carriers. Four-fifths of males with the full mutation are clinically affected, while only half the females with the full mutation are affected. In females with the premutation, the CGG trinucleotide expansion in the FMR-1 gene is hereditarily unstable. There is, therefore, a high risk of the premutation expanding to a full mutation when it is transmitted from a woman to her children. However, when the premutation is passed through men (also known as carrier males or normal-transmitting males), it does not significantly increase in size. The sons of an unaffected man do not receive the X chromosome and so are neither affected nor carriers. His daughters, on the other hand, always receive the premutation and are all unaffected carriers, although their own children are at risk of inheriting the full mutation. Heterozygous females who receive the full mutation from their mothers may have physical and psychological features of fragile-X syndrome. The transmission of the mutation through phenotypically normal daughters to their grandchildren, occurs when the disease severity increases through successive generations. About 30-35% of female carriers have intellectual disabilities, and they are more likely to have similarly affected offspring than are intellectually normal carrier females.

Genetic counselling aims to educate families about the syndrome, its implications and prognosis, supporting them in making informed decisions about the future and in dealing with the emotional impact of the diagnosis. Counselling also identifies others who might need to be alerted about the diagnosis and the availability of testing.

FIGURE 3: Common physical, behavioural and developmental features of fragile-X syndrome Physical features Behavioural Features Developmental Features Broad forehead Elongated face Large, prominent ears Strabismus High arched palate Malocclusion of teeth Hand calluses (due to self-injury) Dermatoglyphics:  radial loops, whorls and arches   A-B ridge count and ulnar loops Pertus excavatum Mitral valve prolapse, cardiomegaly, hypoplasia and dilation of aorta, post-ductal coarctation Macro-orchidism Soft, fleshy skin Scoliosis, pes planus, joint laxity and hyperextensible joints  Brain weight  Size of fourth ventricle  Hippocampal volume  Volume of superior temporal gyrus  Posterior cerebellar vermis Epileptic seizures (25%) (usually generalised tonic-clonic which respond to carbamazepine - half disappear by age 20) Attention deficit hyperactivity disorder Short attention span Impulsivity Enuresis, encopresis Autistic-like features: Gaze aversion (especially to strangers) Social anxiety & shyness Hand-flapping and hand biting Sensory defensiveness (aversion to loud noise, touch, strong smells or eye contact) Poor adaptation to changes in routine   Psychiatric disorders Increased incidence of familial bipolar affective disorders Perseverative mumbling and stereotypic behaviours mask psychosis Psychosis in association with epileptic seizures Mood instability with aggression and depression (particularly in adolescence) Premutation females show increased incidence of schizotypal features and depression Intellectual disabilities: 80% males 50% females Mild to moderate (children) Moderate to severe (adults) Gradual decline in IQ as they grow older but adaptive functioning can be improved Specific cognitive profiles; difficulties in sequential processing, short-term memory deficits and weakness in arithmetic. Fine and gross motor delay Problems with co-ordination Speech abnormalities: Delayed and distorted speech and language (2 years for words, 3 years for short sentences) Tachyphemia Tachylalia Perseveration and delayed echolalia Cluttering of speech

Phenotype
The common physical, behavioural and developmental features of fragile-X syndrome are shown in Figure 3. The somatic phenotype is well formed and easily distinguishable in adults (Figure 4). In children, the behavioural phenotype is more prominent as the physical features are still being formed.

• In typical fragile-X males, the face is long with large, everted ears and a prominent jaw. The forehead is large and quadrangular, with relative macrocephaly.
• Macro-orchidism is almost invariable in DNA-confirmed post-pubertal males. This phenotype has also been described in patients with acquired central nervous system lesions, patients with no abnormality of the FMR-1 gene, and it sometimes co-occurs in Klinefelter, Prader-Willi, Sotos, Rubenstein-Taybi and Down's syndromes.
• Ophthalmological findings include strabismus (40%), high myopia and nystagmus. Musculoskeletal manifestations such as pes planus (flat feet), excessive joint laxity with hyperextensible metacarpophalangeal joints and scoliosis are common. The skin is usually soft and smooth, but there may be calluses from hand-biting. Cardiac abnormalities include mitral valve prolapse and aortic root dilatation, hypoplasia of the aorta and post-ductal coarctation. These are thought to develop during late childhood and adolescence, as in the general population. Taken together, these findings may suggest an underlying connective tissue dysplasia.
• Pregnancy is usually unremarkable and affected babies are normally born at full term. The failure to achieve normal developmental milestones may first alert parents to intellectual handicap. Macro-orchidism is rare before puberty, but the phenotype is more evident as the child grows. In children, it is the behavioural features such as hand-flapping, hand-biting, tactile defensiveness, poor eye contact, hyperactivity, shyness and social anxiety, and perseverative speech that are more notable. Most boys have attentional problems and hyperactivity.
• Affected females usually resemble affected males, though with enlarged ovaries. Unaffected (premutation) females have high rates of premature ovarian failure and dizygous twinning.

FIGURE 3: Common physical, behavioural and developmental features of fragile-X syndrome. Click here

Dermatoglyphic patterns: findings include an increased frequency of radial loops, whorls and arches on the fingertips, abnormal and plantar creases, and a decrease of ulnar loops and ridge counts.

Cognitive functioning

Males - although the level of severity of intellectual disability among boys is equally distributed between mild and severe, the majority of men with fragile-X syndrome have moderate-to-severe degrees of intellectual disability. Specific cognitive profiles show that they have particular difficulties in sequential processing, with short-term memory deficits manifesting as a weakness in arithmetic and visuospatial skills. In spite of the reported decline in IQ test scores with age, adaptive behaviour improves with appropriate training. Although there is no correlation between the size of the CGG repeat (within the full mutation range) and degree of intellectual impairment in males, lower expression of FMRP is thought to correlate with IQ in mosaic males, males with a partially methylated full mutation and full-mutation females.

Females - 50% of female carriers have cognitive impairment. The remaining 50% may demonstrate below-average intellectual functioning (IQ 70-100), specific learning difficulties, and/or psycho­social problems. Affected females without intellectual disability may have specific deficits in the areas of attention, visuo-spatial skills, shyness and social anxiety, and executive functions. The less serious effects in females may be due to the fact that they have two X chromosomes, of which only one is active in each cell. This increases the chances a normal FMR-1 gene owing to random X chromosome inactivation.

Speech and language delay is an early symptom, with first words appearing at about 2 years and short sentences at 3 years. Some children do not develop speech at all. Language ability may be appropriate for an individual's cognitive level, but others often display 'jocular litanic phraseology' with echolalia and speech dysfluency, or 'cluttering'.

FIGURE 4: Men and boys with fragile-X syndrome, showing classical facial features.

Epilepsy and EEG findings
Seizures are the most common neurological finding. They are usually generalized and occur in one-fifth of affected people, usually in the first 15 years of life. They respond well to anticonvulsants, particularly carbamazepine, and half of them disappear by age 20. The most common abnormal EEG findings are rhythmic theta activity, and slowing of background activity. Initial reports of characteristic sleep EEG patterns, such as quasi-rhythmic temporal lobe spikes of medium/high voltage, have not been replicated.

Pathological and neuropathological findings
Pathological findings include abnormal mitral valves with mucoid degeneration and excess mucopolysaccharide; ventricular hypertrophy; cardiac enlargement; and interstitial cell hyperplasia, which causes megalo-testes.
Neuropathological findings include increased brain weight; mildly dilated cerebral ventricles; and loss of Purkinje cells in the cerebellum (which normally show high expression of FMRP).
Neuroimaging studies confirm the dilated cerebral ventricles, and show a decrease in size of the posterior cerebellar vermis.

Behavioural phenotype
In ICD-10, fragile-X syndrome is noted as one of the medical conditions associated with pervasive developmental disorder (PDD). Only a minority of males with fragile-X syndrome have autism, although autism-like behavioural features are seen in almost all people with fragile-X syndrome. The majority show the characteristic profile of social anxiety, gaze aversion to strangers, vocal perseveration, delayed echolalia, and stereotypies such as hand-biting and hand-flapping. Their greeting behaviour is unusual and may involve turning their whole body away when greeting others. The prevalence of fragile-X syndrome in autism is 1.6%, which is lower than previous estimates.

Associated psychiatric disorders
Attentional deficits are more common in fragile-X syndrome than in other causes of intellectual disability. Inattentiveness, restlessness and fidgetiness tend to persist over time. Premutation carriers have increased social anxiety and mood lability. It may be difficult to distinguish delusions and hallucinations from the usual fragile-X behaviour, which may include perseverative mumbling and stereotypies. In female carriers, schizotypal features are more common in mildly affected people and fragile-X positive heterozygotes, and depression in fragile-X negative heterozygotes. The occurrence of comorbid bipolar affective disorder in other family members has been described.

Management
While there is no cure for fragile-X syndrome, many areas of intervention can improve the lives of those affected and their families. All affected people can make progress with proper education, therapy and support. A multidisciplinary approach is necessary to manage the multifaceted problems encountered. Each child should be formally assessed to establish his or her needs. Speech therapists, behavioural therapists, special educators and paediatricians are all likely to be involved.

The early years are of vital importance for stimulating maximum learning in children with the syndrome, and intervention at this stage can prevent many problems later. Services that can be offered include family training to encourage physical, speech and sensory training, and the promotion of a routine for the child, which helps to alleviate anxiety.

Family education and counselling is essential to facilitate parents' acceptance and understanding of the child and to encourage patience and persistence with a child who may seem uncooperative. Daily living skills, which include eating, sleeping and personal hygiene and toilet training, can be challenging for children and adults with fragile-X syndrome and this form of developmental delay without support can be a long and frustrating experience for the family.

Managing difficult behaviour - aggressive behaviour occurs in 20-30% of individuals with fragile-X syndrome. It is related to problems with impulsivity, over-reactivity to stimuli, high anxiety levels, adverse reactions to changes in routine, and mood instability. Individuals may be described as violent and unpredictable but their behaviour is often misunderstood and may represent a desire to be left alone or to escape a threatening situation rather than malicious intent. Concomitant use of behaviour modification with counselling and psychotropic medication can be beneficial in managing aggression. Stimulants such as methylphenidate, and other medications such as clonidine may be effective if the child has attention deficit hyperactivity disorder (ADHD) or if the aggression stems from impulsive behaviour. Alternatively, selective serotonin reuptake inhibitors (SSRIs) can be considered if excess anxiety, obsessive-compulsive features or depression are causes of their aggressive behaviour.

Special needs education should provide structured activities suitable for children with a short attention span, and there should be minimal auditory and visual distractions in the classroom. Teaching support often emphasizes face-to-face contact, yet individuals with fragile-X syndrome often find gaze contact highly aversive.

Strengths and weaknesses - most children with the syndrome have good imitation skills and functional life skills once acquired. Their weaknesses are poor auditory short-term memory, poor sequential processing, difficulties with numeracy and visuo-spatial skills and abstract thinking. A teaching plan must be devised to optimize a child's stronger areas without ignoring those that may be problematic and so require special attention.

Managing associated medical conditions - cardiac functioning needs to be assessed because of the increased incidence of mitral valve prolapse and cardiomegaly. Seizure disorders usually respond well to anticonvulsants such as carbamazepine and sodium valproate. Antipsychotics are indicated for psychotic symptoms, but care must be taken in those with comorbid epileptic seizures. SSRIs are usually reserved for comorbid depression and overwhelming obsessive-compulsive problems.

The future
Increasing awareness of the condition has resulted in the growth of national and international fragile-X societies. However, many families remain undiagnosed. Most of the features discussed in this contribution were described on cytogenetically-confirmed cases. The advent of newer tests has brought with it the need to revisit some of these features and to re-estimate the true prevalence of fragile-X syndrome. Although the exact function of FMRP is not yet known, the mapping of the human genome has paved the way for possible gene therapy or the more feasible protein replacement approaches. More pieces of the genetic puzzle are emerging, such as recent reports of Parkinson-like neurological symptoms among grandfathers of individuals with fragile-X syndrome. Finally, the use of the FMRP antibody test promises to revolutionize screening and improve case detection.

REFERENCES
Dykens E M, Hodapp R M, Leckman J F. Behaviour and Development in Fragile X Syndrome. London: Sage, 1994
Hagerman R J, Cronister A. Fragile-X Syndrome: Diagnosis, Treatment, and Research. 2nd edition. Baltimore, MD: Johns Hopkins University Press, 1996.
Lubs H. A marker X chromosome. Am J Hum Genet 1969; 21: 231-44.
Pembrey M E, Barnicoat A J, Carmichael B, Bobrow M, Turner G. An assessment of screening strategies for fragile X syndrome in the UK. Health Technol Assess 2001; 5: 1-95.
Sabaratnam M, Tony J. Fragile-X syndrome. Irish Psychiatrist 2002; 3: 46-52.
Yu S, Pritchard M, Kremer E et al. Fragile X genotype characterised by an unstable region of DNA. Science 1991; 252: 1179-81.

USEFUL ADDRESS
The Fragile X Society,
Rood End House,
6 Stortford Road,
Great Dunmow,
Essex CM6 1DA

Tel: 01371 875100
Website: www.fragilex.org.uk

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