Fragile X Syndrome

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Fragile X syndrome is the most common form of heritable mental retardation affecting approximately 1 in 4000 males and 1 in 8000 females. Martin and Bell first documented X-linked mental retardation in 1943. Subsequent identification of a fragile site on the long arm of the X chromosome (Lubs, 1969), dis- covery of cell culture medium-dependent fragile site, and recognition of a unique constellation of physical features served to distinguish fragile X syndrome from other X-linked mental retardation syndromes.

In 1991, Verkerk et al. identified a single gene that was associated with symptoms of the disorder. The gene, known as frag- ile X mental retardation gene 1 (FMR1), exhibited a novel form of mutation, a sequence of three nucleotides (CGG) that was repeated many times in patients with fragile X syndrome.

GENETICS/BASIC DEFECTS

1. Inheritance not conforming to usual rules governing X- linked traits

a. Presence of asymptomatic transmitting males b. Presence of affected female carriers who inherited

the gene

2. Caused by mutations in a trinucleotide repeat expansion (CGG) found in the coding sequence of the FMR1 gene, mapped to Xq27.3

a. Expanded CGG repeats >200 (full mutation): associated with fragile X syndrome

b. High repeat number (full mutation) leads to hypermethylation of an upstream promoter region and subse- quent silencing (inactivating) of the FMR1 gene c. A small expansion (premutation) with approximately

50–200 CGG repeats: usually not associated with cognitive deficits

d. Expansion to a full mutation from the premutation in normal carriers occurs only when it is transmitted by a female

e. Presence of anticipation phenomenon 3. The Sherman paradox

a. A perplexing and confusing fragile X pedigree pattern was discovered during mid-1980s by Sherman et al. who discovered the large discrepancy in risks for mental retardation within fragile X families con- taining transmitting males. This phenomenon was termed the “Sherman paradox” by Opitz (1986)

i. 20% of males within fragile X families who carried the mutation are unaffected clinically and intellectually and became known as transmitting males

ii. An increasing risk of mental retardation in grandsons of normal transmitting males: a special form of genetic anticipation (the increase in disease severity through successive generations)

iii. About a third of obligate carrier females are mentally impaired

iv. More than half of known carrier females are either mildly impaired or expressed the fragile site on one of their X chromosomes

b. Possible explanations of the Sherman paradox i. An unaffected transmitting male who inherits a

premutation from his unaffected carrier mother, who has 50 to 200 repeats but probably a number closer to the lower end of the premutation range ii. Offspring of a carrier mother with the number of

repeats still within the premutation range, even if amplification occurs during oogenesis

iii. Transmission of the premutation to the daughters by the transmitting male. No amplification has occurred during spermatogenesis; hence the daughters also have a premutation and are unaffected iv. Amplification to more than 200 repeats occurs

during oogenesis of the transmitting male’s daughters whose sons will receive a full mutation and be affected and their daughters may or may not be affected

4. Genotypic–phenotypic correlations

a. Fragile X full mutation and mental retardation with a wide spectrum of phenotypic effects in both sexes

i. Mental retardation in 100% of males

ii. Usually a milder form of retardation in 60% of females

b. Large CGG amplification

i. Associated with hypermethylation

ii. Almost invariably correlated with the most severe fragile X phenotype

iii. Seen in males with the full mutation pattern c. Premutation expansions that are not usually accom-

panied by methylation: minimal or no neurologic repercussion

d. Correlation of methylation in FMR1 expression and cognitive function

i. Individuals with full mutation without methylation scored better than those with full mutation and complete methylation

ii. Individuals with full mutation and partial methylation showed intermediate values

e. Presence of the FMR1 protein (FMRP) responsible for:

i. Lack of neurologic abnormalities in individuals with premutation

ii. Milder phenotype of males having full mutation without full methylation

5. Pathogenesis

a. A consequence of the absence or deficit of the FMRP b. Absence of FMRP in the brain: the likely cause of mental retardation in patients with fragile X syndrome

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CLINICAL FEATURES

1. Affected males a. CNS involvement

i. Delayed developmental milestones ii. Mild to severe mental retardation

iii. Difficulty with abstract thinking, sequential processing, mathematics, short-term memory, and visual motor coordination

iv. Seizures

b. Connective tissue dysplasia i. Hyperextensible finger joints ii. Double-jointed thumbs iii. Flat feet

iv. High-arched palate

v. Mitral valve prolapse (55%, diagnosed by echocardiography)

vi. Dilatation of the ascending aorta vii. Inguinal hernia

viii. Soft and velvet-like skin c. Typical facial features

i. Long face

ii. Prominent forehead iii. Prominent/long ears

iv. Prominent jaw d. Other features

i. Macroorchidism present in over 80% of adult fra(X) males

ii. Pectus excavatum iii. Scoliosis

iv. Strabismus

v. Recurrent otitis media in early childhood vi. Reproduction documented but rare because of

significant mental retardation e. Behavior abnormalities

i. Stereotyped with odd mannerisms (hand flap- ping/biting)

ii. Tactile defensiveness

iii. Poor eye contact (excessive shyness) iv. Attention-deficit/hyperactivity disorder

a) Hyperactivity b) Temper tantrums c) Distractibility d) Mood lability v. Speech disorder

a) Perseveration b) Litany speech c) Echolalia vi. Autism

vii. Autistic-like features

viii. Schizotypal personality disorder ix. Anxiety disorder

2. Females heterozygous for full mutation alleles

a. 50% with cognitive deficits with learning disabilities, borderline IQ, or mental retardation

b. 50% with normal intellectual function

3. Females heterozygous for premutation alleles (carriers) at risk for premature ovarian failure (early onset of menopause before age 40 years)

DIAGNOSTIC INVESTIGATIONS

1. Pedigree analysis

2. Karyotyping of cells grown in folate- or thymidine- depleted cell culture media

a. A “fragile” site on one of the X chromosomes (Xq27.3) that appeared as a constriction on the distal long arm in many patients

b. Cells exhibiting fragile X chromosome: 5–50%

c. Cytogenetic studies now rendered obsolete by direct DNA testing

3. Indications for molecular genetic testing

a. Individuals of either sex with mental retardation, developmental delay, or autism

i. Any physical or behavioral characteristics of fragile X syndrome

ii. A family history of fragile X syndrome

iii. Male or female relatives with undiagnosed mental retardation

b. Individuals seeking reproductive counseling who have a family history of fragile X syndrome or undiagnosed mental retardation

c. Fetus of a known carrier mother

d. Patients with cytogenetic fragile X test result discor- dant with phenotype

i. Patients with a strong clinical impression of being affected with or carrier of fragile X syndrome but had a negative or ambiguous cytogenetic test result

ii. Patients with an atypical phenotype of fragile X syndrome but had a positive cytogenetic test result

4. Direct DNA analysis for point mutation or deletion in FMR1 gene. Mutation analysis to determine the CGG repeat size by Southern blot hybridization and poly- merase chain reaction (PCR): mutation detection rate

>99% (commercially available)

5. Methylation analysis by Southern blot analysis to deter- mine the FMR1 methylation status

6. Types of FMR1 repeat expansion mutations a. Normal: 5–50 CGG repeats

b. Premutation

i. 51–200 CGG repeats

ii. Methylation status of FMR1: premutation alleles are usually unmethylated and FMRP production is normal. Therefore, individuals with permutation are clinically unaffected

iii. Males: clinically unaffected iv. Females: clinically unaffected c. Full mutation

i. >200 CGG repeats. Expansion of the repeat more than 200 generally results in hypermethylation of both the CpG island and the CGG repeat within the FMR1 gene

ii. Methylation status of FMR1: completely methylated

iii. Males: affected

iv. Females: affected in about 50% of cases; unaffected in about 50% of cases

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d. Mosaicism

i. Number of CGG repeats varies between premutation and full mutation in different cell lines ii. Methylation status of FMR1: partially methylated

(unmethylated in the premutation cell line and methylated in the full mutation cell line) iii. Males: affected but may function higher than

individuals with full mutation

iv. Females: highly variable clinical expression ranging from normal intellect to affected e. Methylation mosaicism

i. Number of CGG repeats >200

ii. Methylation status of FMR1 gene: partially methylated (mixture of methylated and unmethylated cell lines)

iii. Males: affected but may function higher than individuals with full mutation

iv. Females: highly variable clinical expression ranging from normal intellect to affected f. Unmethylated full mutation

i. Number of CGG repeats >200

ii. Methylation status of FMR1 gene: unmethylated iii. Males: nearly all are affected but may have high functioning mental retardation to low normal intellect

iv. Females: highly variable clinical expression ranging from normal intellect to affected 7. Immunocytochemical tests based on the direct detection

of FMRP using monospecific antibodies. This FMRP detection assay is based on the presence of FMRP in cells from unaffected individuals and its absence in cells from patients with fragile X syndrome. This assay is proven to be a reliable alternative method to identify male patients with fragile X syndrome. The new test on hair roots is suitable for use in large screening programs among males.

The immunocytochemical tests can also be used in patients with mosaic pattern, affected premutation males, and intragenic mutations.

GENETIC COUNSELING

1. Recurrence risk: adequate genetic counseling depends on accurate diagnosis at the molecular level

a. Individuals identified as non-carriers: the zero risk of transmitting fragile X syndrome to the next generation b. Individuals identified as carriers: the risk of having children with fragile X syndrome depending on the sex of the carrier parent, the sex of the child, and the size of the CGG repeats

i. Premutation carrier males: considered “transmitting males”. All daughters of transmitting males are unaffected premutation carriers. However, the grandsons and granddaughters of a transmitting male are at risk for developing fragile X syndrome ii. All mothers of a child with FMR1 full mutation (expansion >200 CGG trinucleotide repeats), considered carriers of an FMR1 gene expansion (either full mutation or premutation and may be affected)

iii. Females with premutation: an increased risk of passing on the full mutation and having offspring with the fragile X phenotype (the fragile X premutation can expand to the full mutation during maternal meiosis)

iv. Carrier males who may reproduce: essentially zero risk of having male offspring with Fragile X syndrome, since all their sons receive their Y chromosome [except in the rare instance of the fragile X chromosome coming into the family from the other source (mother)]. All the daughters of male carriers will have premutations regardless of the size of paternal amplifications and will not have fragile X syndrome. Only a few reports with premutation males having full mutation daughters

c. Females with full mutation: Her offspring, if they inherit the fragile X locus, all will have full mutation

i. All her sons will have fragile X syndrome ii. About 50–60% of her daughters will have fragile

X syndrome, exceeding the 35% applicable to all female carriers of the fragile X chromosome.

Affected females generally have less severe intellectual disability than found in affected males

d. Males with full mutations i. Mentally retarded

ii. Generally do not reproduce

2. Risks to pregnant women with an expanded FMR1 gene a. A repeat size of 40–59: safe (0% risk) with no fetal

full mutations

b. A repeat size of 60–80: low risk (14%) of full mutation in the fetus

c. A repeat size of over 80–100: a significant increase in risk (89%) of developing full mutation in the fetus d. A repeat size of 100–200: 100% risk of developing

full mutation in the fetus

3. Prenatal diagnosis by amniocentesis or CVS

a. Prenatal diagnosis using direct DNA analysis to women discovered to be premutation carriers b. The limitation: inability to accurately predict pheno-

type in female fetuses with full mutation

c. CVS: follow-up amniocentesis or testing using PCR, necessary to determine the size of the FMR1 alleles in a methylation-independent manner

4. Management

a. Early intervention programs for developmental delay including speech and language therapies, physical therapy, and occupational therapy

b. Behavioral interventions including psychopharmaco- logical therapy

c. Special educations d. Vocational planning

e. Anticonvulsants for seizure control

f. Prophylactic antibiotics for surgical or dental proce- dures in patients with mitral valve prolapse

g. Orthopedic care for joint dislocations, flat feet, and scoliosis

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Fig. 1. Two brothers with fragile X syndrome showing large ears, accompanied by their mother.

Fig. 2. A pair of brothers with fragile X syndrome showing a long face with large ears.

Fig. 3. Another pair of male siblings with fragile X syndrome.

Fig. 4. A chromosome spread showing a fragile X chromosome (arrow).

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Fig. 5. A large family affected by fragile X syndrome. The first boy (A, age 15) is the most affected one with CGG repeats of >1500. He was not toilet trained until age 5 or 6 years. He never spoke until after auditory training. He may watch TV for 6–8 hours straight and get agi- tated if someone approaches him. The brother (B, age 10) and sister (C, age 16) have CGG repeats of 1200 and 600 respectively. Another sister (D) is a carrier with CGG repeats of 110 who has 4 children. The daughter (age 3, the first one from the left) has CGG repeats of 700.

She has a long face with prominent ears, a high-arched palate and flat feet. She suffers from hyperactivity, tactile/oral/olfactory defensiveness, gaze aversion, poor postural alignment, food cramming, hand biting, excessive drooling, poor self-regulation, and speech difficulties including echolalia. The other 3 children are normal with normal CGG repeats. The maternal grandmother has CGG repeats of 126.

Fig. 6. A 66-year-old male with fragile X syndrome. The patient has mental retardation, a long face with large ears, and macroorchidism.

Molecular analysis revealed CGG repeats of about 400.

Fig. 7. A 46-year-old male with methylation mosaic fragile X syn- drome (mixture of methylated full mutation allele of approximately 450 repeats in some cells and unmethylated premutation size allele of approximately 200 repeats in some cells). He is tall and has mental retardation, a long narrow face, slightly large ears, and macroorchidism.