Fragile X Trinucleotide Repeat Sizing

Background

Fragile X syndrome is the most common form of inherited mental retardation and the second most frequent cause of mental retardation after Down syndrome. The disorder is inherited in an X-linked dominant manner, with 30% of males carrying the fragile X chromosome being phenotypically normal. For females with a full mutation, 50% will have cognitive impairment and 50% will be of normal intellect. It is typically caused by expansion of a CGG repeat region in the 5' UTR of the FMR1 gene and is rarely caused by deletions or point mutations within this gene. This lengthening is known to induce the methylation of a CpG island just upstream from the repeat region. The resulting methylation then causes the down regulation of FMR-1 protein expression and is manifested as Fragile X syndrome.

The length of the CGG repeat in the normal population is polymorphic and ranges between 5 and 44 repeats, with a repeat number of 29 or 30 being the most common. Premutation carrier females and transmitting males show no phenotypic effect and range between 56 and 200 repeats. Accurate determination of CGG repeat numbers is essential for appropriate risk assessment and counseling of premutation carriers. Patients possessing premutation alleles (56-200 CGG repeats) were once thought to be asymptomatic. Recent work has shown that individuals with premutations can manifest Fragile X associated tremor/ataxia syndrome (FXTAS), premature ovarian failure, or mild cognitive/behavioral defects. The number of repeats correlates to the severity of disease. In a full mutation, there are over 200 repeats and the CpG island is methylated. AGG interruptions of the CGG repeat domain are thought to lead to a lowered risk of CGG expansion in the next generation.

OMIM: 300624

Clinical Utility

Detection of expansions in FMR1 that affect the severity of Fragile X syndrome. The test is done on peripheral blood specimens for confirmation of clinical diagnosis, carrier status or presymptomatic testing.

Methodology

A PCR assay is performed by utilizing a set of primers that encompass the Fragile X CGG repeat region. The product size is determined using a size standard, and CGG repeat number is calculated. In addition to the sizing primers an additional primer consisting of 5 CGG repeats is used to produce a stutter plot that allows confirmation of total repeat size and indicates the presence or absence of AGG interruptions within the CGG repeat domain.

Loci Tested

FMR1 5'UTR

Specimen Requirements

Turnaround Time

Within 3-10 business days of receipt

CPT

Shipment Must Include

References

1. Hagerman, P.J and R.J. Hagerman. 2004. The fragile X premutation: a maturing perspective. Am. J.Hum. Genet. 74: 805-816.
2. Hundscheid, R.D., A.P. Smits, C.M. Thomas, L.A. Kiemeney and D.D. Braat. 2003. Female carriers of fragile X premutations have no increased risk for additional diseases other than premature ovarian failure.. Am. J. Med. Genet. A. 117: 6-9.
3. Jacquemont, S., et al. 2003. Fragile X premutation tremor/ataxia syndrome: molecular, clinical and neuroimaging correlates. Am. J. Hum. Genet. 72: 869-878.
4. Jacquemont, S. M.A. Leehey, R.J. Hagerman, L.A. Beckett and P.J. Hagerman. 2006. Size bias of fragile X premutation alleles in late-onset movement disorders. J. Med. Genet. 43: 804-809.
5. Sullivan, A.K. 2005. Association of FMR1 repeat size with ovarian dysfunction. Hum. Reprod. 20: 402-412.
6. Ying-Hue Fue, et al (1991) Variation of the CGG Repeat at the Fragile X Site Results in Genetic Instability; Resolution of the Sherman Paradox, Cell, 67: 1047-1058.
7. Baskaran N, et al (1996) Uniform Amplification of a Mixture of Deoxyribonucleic Acids with Varying GC Content, Genome Methods, 6: 633:638.
8. Papp A.C., et al (1996) Strategies for Amplification of Trinucleotide Repeats: Optimization of Fragile X and Androgren Receptor PCR, Molecular Diagnosis 1(1): 59-64