Final Diagnosis -- Fragile X Syndrome

DIAGNOSIS

This male patient shows size mosaicism with a full mutation FMR1 allele (>200 CGG repeats) and a premutation FMR1 allele with 86 repeats (c.-129CGG[86]).

BACKGROUND Fragile X syndrome is the most common known form of inherited mental retardation, with a frequency of 1 in 1550 males and 1 in 2500 females. It was first described by Martin and Bell in 1943 to have sex-linked inheritance and a more severe phenotype in boys. Clinical features include delays in motor skills development, delays in language development, autism spectrum disorder, hyperactivity, anxiety, and moderate to severe intellectual disabilities. During and after puberty, affected individuals classically manifest a long and narrow face, large ears, hypermobile joints, and macroorchism in males. Girls with Fragile X Syndrome have similar mental and physical features to boys, although, less severe and at lower rates [1-4].

GENOMICS This X-linked disorder is associated with a disease-causing expansion of CGG trinucleotide units in the 5' untranslated region of the familial mental retardation-1 (FMR1) gene with locus Xq27.3. The FMR1 gene encodes a RNA binding protein involved in the regulation of neural mRNA stability and translation, which are important in brain development. Four different types of alleles with different clinical manifestations are described depending on the number of CGG trinucleotide repeats. Expansion of the number of CGG repeats to the full mutation range allele (>200 repeats) leads to hypermethylation of the FMR1 gene promoter and ultimately transcriptional silencing. The premutation allele (56-200 CGG repeats) is not typically associated with methylation of the FMR gene promoter. However, premutation alleles are unstable and can lead to trinucleotide expansion during maternal transmission. The allele can thus transition from a premutation to a full mutation allele, with odds depending on several factors including the range of repeats in the patient, number of AGG interruptions, and age of the mother. Gray zone alleles (45-55) show mild instability and are precursors to premutation alleles, but they do not expand to a full mutation during oogenesis. Lastly, normal alleles contain up to 44 CGG repeats and are not associated with an increased risk of expansion in future generations [5-10].

METHODS

After genomic DNA isolation from peripheral blood leukocytes and enzymatic digestion, the Fragile X locus in this patient was sized by triplet repeat (TRP) PCR amplification followed by high-throughput capillary electrophoresis. In TRP PCR, the forward PCR primer is located upstream of the FMR1 CGG region while the fluorescently labeled reverse primers overlap the CGG repeats and adjacent non-repeat sequence. Multiple PCR products are produced, creating a ladder of amplicons on electrophoresis. Conventional PCR amplification of CG-rich regions is difficult, which becomes even more challenging with trying to detect increasing numbers of CGG repeats. TRP PCR amplification increases the amount of full-length trinucleotide repeat products and circumvents preferential amplification of normal alleles in patients with multiple alleles, such as in females and mosaics [7-10].

DISCUSSION

Expanded CGG trinucleotide units in the 5' untranslated region of the FMR1 allele can lead to instability and lead to size mosaicism. This refers to an individual with subpopulation of cells with different CGG repeat allele sizes, such as one population with a full mutation and another population with a premutation. This is a relatively common phenomenon with an estimated frequency between 10-40%. In addition to size mosaicism, variation in methylation status of full mutations can occur. Mosaicism has been shown to affect penetrance of the disorder and phenotype.

In our example (Figure 1), the patient shows size mosaicism with a full mutation FMR1 allele (200 CGG repeats) and premutation FMR1 allele (86 CGG repeats). This result is due to a deletion in the CGG region and flanking sequences of the FMR1 allele in a subpopulation of cells with full mutation alleles that were expanded during meiosis. The mechanism leading to CGG instability and deletion is still not fully understood, but is thought to involve DNA polymerase slippage and strand mis-pairing leading to CGG length reduction. Studies have also shown CpG methylation in the FMR1 allele may be more susceptible to deletions [11-15].

The identification of the full mutation in this male is diagnostic of Fragile X syndrome. The patient's mother is highly likely to be an obligate carrier of a premutation or full mutation allele. Moreover, the behavioral issues and intellectual disorders seen in the maternal uncle would be worth investigating for FMR1 mutational analysis. There is currently no cure for Fragile X syndrome, but early diagnosis and intervention can help this patient significantly improve his quality of life.

REFERENCES

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12. Schmucker B, Ballhausen WG, Pfeiffer RA. Mosaicism of a microdeletion of 486 bp involving the CGG repeat of the FMR1 gene due to misalignment of GTT tandem repeats at chi-like elements flanking both breakpoints and a full mutation. Hum Genet. 1996;98(4):409-414.
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Contributed by Daniel Geisler, MD and Tim D. Oury, MD, PhD