• Triplet repeat expansion
• Somatic mosaicism
• Sex-specific anticipation
• DNA methylation
• Haplotype effect
Major Phenotypic Features
• Age at onset: Childhood
• Intellectual disability
• Dysmorphic facies
• Male postpubertal macroorchidism
History and Physical Findings
R.L., a 6-year-old boy, was referred to the developmental pediatrics clinic for evaluation of intellectual disability and hyperactivity. He had failed kindergarten because he was disruptive, was unable to attend to tasks, and had poor speech and motor skills. His development was delayed, but he had not lost developmental milestones: he sat by 10 to 11 months, walked by 20 months, and spoke two or three clear words by 24 months. He had otherwise been in good health. His mother and maternal aunt had mild childhood learning disabilities, and a maternal uncle was intellectually disabled. The findings from his physical examination were normal except for hyperactivity. The physician recommended several tests, including a chromosomal microarray, thyroid function studies, and DNA analysis for fragile X syndrome. Diagnostic analysis of the FMR1 gene was consistent with fragile X syndrome.
Disease Etiology and Incidence
Fragile X syndrome (MIM 300624) is an X-linked disorder of intellectual disability that is caused by mutations in the FMR1 gene on Xq27.3 (see Chapter 12). It has an estimated prevalence of 16 to 25 per 100,000 in the general male population and half that in the general female population. The disorder accounts for 3% to 6% of intellectual disability among boys with a positive family history of cognitive deficits and no birth defects.
The FMR1 gene product, FMRP, is expressed in many cell types but most abundantly in neurons. The FMRP protein may chaperone a subclass of mRNAs from the nucleus to the translational machinery.
More than 99% of FMR1 mutations are expansions of a (CGG)n repeat sequence in the 5′ untranslated region of the gene (see Chapter 12). In normal alleles of FMR1, the number of CGG repeats ranges from 6 to approximately 50. In disease-causing alleles or full mutations, the number of repeats is more than 200. Alleles with more than 200 CGG repeats usually have hypermethylation of the CGG repeat sequence and the adjacent FMR1 promoter (Fig. C-17). Hypermethylation epigenetically inactivates the FMR1 promoter, causing a loss of FMRP expression.
FIGURE C-17 Polymerase chain reaction (PCR) analysis of FMR1 CGG repeat number in a normal male (A), a premutation female (B), and a full mutation female (C). The number of CGG repeats is on the x-axis, and fluorescence intensity is on the Y-axis. Normal and premutation ranges are boxed in gray; full mutation range is boxed in pink with characteristic stutter from the repeat-targeted primer in the gray box. D, Agarose gel separation of expanded alleles after PCR with FMR1-specific primers. Lane 1, size markers; Lane 2, full mutation male mosaic for approximately 280 and approximately 350 CGG alleles; Lanes 3 and 4, normal males; Lane 5, full mutation male. E, Methylation-sensitive PCR determines methylation status of alleles near the premutation/full mutation boundary in males; positions of methylated and unmethylated alleles are indicated. Lane 1, size markers; Lane 2, abnormal male with mosaic methylation with approximately 140, approximately 350, and approximately 770 CGG alleles; Lane 3,male with expanded unmethylated repeat in the premutation range; Lane 4, affected male with complete methylation; Lane 5, male with mosaic methylation. Note that methylation status in females can only be determined by Southern blot (see Fig. 7-22). See Sources & Acknowledgments.
FMR1 full mutations arise from premutation alleles (approximately 59 to 200 CGG repeats) with maternal transmission of a mutant FMR1 allele but not with paternal transmission; in fact, premutations often shorten with paternal transmission. Full mutations do not arise from normal alleles. Because the length of an unstable CGG repeat increases each generation when it is transmitted by a female, increasing numbers of affected offspring are usually observed in later generations of an affected family; this phenomenon is referred to as genetic anticipation (see Chapter 7).
The risk for premutation expansion to a full mutation increases as the repeat length of the premutation increases (see Fig. 7-23). Not all premutations, however, are equally predisposed to expand. Although premutations are relatively common, progression to a full mutation has been observed only on a limited number of haplotypes; that is, there is a haplotype predisposition to expansion. This haplotype predisposition may relate partly to the presence of a few AGG triplets embedded within the string of CGG repeats; these AGG triplets appear to inhibit expansion of the string of CGG repeats, and their absence in some haplotypes therefore may predispose to expansion.
Phenotype and Natural History
Fragile X syndrome causes moderate intellectual disability in affected males and mild intellectual deficits in affected females. Most affected individuals also have behavioral abnormalities, including hyperactivity, hand flapping or biting, temper tantrums, poor eye contact, and autistic features. The physical features of males vary in relation to puberty such that before puberty, they have somewhat large heads but few other distinctive features; after puberty, they frequently have more distinctive features (long face with prominent jaw and forehead, large ears, and macro-orchidism). Because these clinical findings are not unique to fragile X syndrome, the diagnosis depends on molecular detection of mutations. Patients with fragile X syndrome have a normal life span.
Nearly all males and 40% to 50% of females who inherit a full mutation will have fragile X syndrome. The severity of the phenotype depends on repeat length mosaicism and repeat methylation (see Fig. C-17). Because full mutations are mitotically unstable, some patients have a mixture of cells with repeat lengths ranging from premutation to full mutation (repeat length mosaicism). All males with repeat length mosaicism are affected but often have higher mental function than those with a full mutation in every cell; females with repeat length mosaicism are normal to fully affected. Similarly, some patients have a mixture of cells, with and without methylation of the CGG repeat (repeat methylation mosaicism). All males with methylation mosaicism are affected but often have higher mental function than those with a hypermethylation in every cell; females with methylation mosaicism are normal to fully affected. Very rarely, patients have a full mutation that is unmethylated in all cells; whether male or female, these patients vary from normal to fully affected. In addition, in females, the phenotype is dependent on the degree of skewing of X chromosome inactivation (see Chapter 6).
Female carriers of premutations (but not full mutations) are at a 20% risk for premature ovarian failure. Male premutation carriers are at risk for the fragile X associated tremor/ataxia syndrome (FXTAS). FXTAS manifests as late-onset, progressive cerebellar ataxia and intention tremor. Affected individuals may also have loss of short-term memory, executive function, and cognition as well as parkinsonism, peripheral neuropathy, lower limb proximal muscle weakness, and autonomic dysfunction. Penetrance of FXTAS is age-dependent, manifesting in 17% in the sixth decade, in 38% in the seventh decade, in 47% in the eighth decade, and in three fourths of those older than 80 years. FXTAS may manifest in some female premutation carriers.
No curative treatments are currently available for fragile X syndrome. Therapy focuses on educational intervention and pharmacological management of the behavioral problems.
The risk that a woman with a premutation will have an affected child is determined by the size of the premutation, the sex of the fetus, and the family history. Empirically, the risk to a premutation carrier of having an affected child can be as high as 50% for each male child and 25% for each female child but depends on the size of the premutation. On the basis of analysis of a relatively small number of carrier mothers, the recurrence risk appears to decline as the premutation decreases from 100 to 59 repeats. Prenatal testing is available by use of fetal DNA derived from chorionic villi or amniocytes.
Questions for Small Group Discussion
1. Discuss haplotype bias in disease; that is, the effect of haplotype on mutation development (fragile X syndrome), disease severity (sickle cell disease), or predisposition to disease (autoimmune diseases).
2. Fragile X syndrome, myotonic dystrophy, Friedreich ataxia, Huntington disease, and several other disorders are caused by expansion of repeat sequences. Contrast the mechanisms or proposed mechanisms by which expansion of the repeat causes disease for each of these disorders. Why do some of these disorders show anticipation, whereas others do not?
3. The sex bias in transmission of FMR1 mutations is believed to arise because FMRP expression is necessary for production of viable sperm. Compare the sex bias in transmitting fragile X syndrome and Huntington disease. Discuss mechanisms that could explain biases in the transmitting sex for various diseases.
4. What family history and diagnostic information are necessary before prenatal diagnosis is undertaken for fragile X syndrome?
5. How would you counsel a pregnant woman carrying a 46,XY fetus with 60 repeats? A 46,XX fetus with 60 repeats? A 46,XX fetus with more than 300 repeats?
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