Xpansion Interpreter™

Features of Fragile X syndrome vary widely but can include mild to severe cognitive delays, seizures, behavioral differences such as perseverative speech and hand flapping, and characteristic facial features such as large ears and a long face (1). About 1 in 4,000 males and 1 in 8,000 females has Fragile X syndrome (2).

The molecular basis for Fragile X syndrome is an increased number of trinucleotide CGG repeats in the 5’ promoter region of the FMR1 gene. FMR1 alleles can be further categorized based on the number of CGG repeats, and premutation alleles may expand from parent to child to a full mutation that can cause features of Fragile X syndrome. In addition, people with premutations may develop Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) and women may develop Fragile X-associated Primary Ovarian Insufficiency (FXPOI).

Table 1: Categories of triplet repeat mutations in the FMR1 gene (1).

AGG interruptions within the CGG repeats of the FMR1 gene have long been suggested to stabilize alleles to protect against expansion (5-8), yet to date, genetic counseling relies upon the total number of CGG repeats. In fact, the number of uninterrupted CGG repeats and number and location of AGGs contribute significantly to the risk for expansion.

Figure 1: Diagram of FMR1 gene and AGG locations.

Asuragen’s scientists collaborated with Fragile X experts at the New York State Institute for Basic Research in Developmental Disabilities, Rush University Medical Center, Emory University School of Medicine, and the UC Davis M.I.N.D. Institute to conduct a large study using the technology behind Xpansion Interpreter to examine CGG repeats and AGG interruptions in 456 mother-to-child transmissions for alleles with 45-69 total repeats. The study showed a clear modification of the probability of expansion, of variable sizes and degrees, when number and location of AGG interruptions and uninterrupted CGG repeats was considered.

Knowing the number of uninterrupted CGGs can help tailor the risk for an allele to expand. Figure 2 can be used as a tool for interpretation (9).

Figure 2: Probability and magnitude of expansion in 456 mother-to-child transmissions based on the number of uninterrupted 3’ CGG repeats within the 5’ UTR of FMR1.

• Small premutation alleles that contain large tracts of uninterrupted alleles face a much higher risk for expansion than would be determined from the total number of repeats (Figures 2 and 3).

• Intermediate alleles that contain AGGs are highly stable (Figure 3), a fact that has also been suggested by reports that the majority of large alleles don’t contain more than 1 AGG (7).

Figure 3: Modification of expansion risk based on number of AGG interruptions.

Data from Figures 2 and 3 were presented at the 2010 AMP and 2011 ACMG annual conferences.

Case 1: Smallest reported expansion to full mutation (10)

A 17-year-old male with features of Fragile X syndrome was tested and revealed to have 538 CGG repeats in FMR1. Family members were tested, and the maternal grandfather had a 52-repeat (CGG)₁₀AGG(CGG)₉AGG(CGG)₃₁ FMR1 genotype, and the mother had 56 uninterrupted CGGs. At that time, the smallest allele that has been reported to expand to a full mutation was a 59-repeat allele that was also uninterrupted.

Small premutation alleles with uninterrupted CGGs (no AGGs) are highly unstable. While most will not expand to a full mutation as the allele in this case, a patient with an uninterrupted small premutation allele certainly faces a much higher risk for expansion than one with AGG interruptions.

Case 2: Reassurance for a female carrier with a 55-repeat allele

For a patient with a 55-repeat allele, the a priori risk for expansion to a full mutation is low; in this case, the risk is about 4% (11). Based on total repeats alone, there is no way to further distinguish higher-risk alleles from the more common stable alleles in this repeat range. In addition, Case 1 above describes a carrier with 56 uninterrupted repeats that expanded to a full mutation in one generation (10).

Table 2: Probability and magnitude of expansion for 36 alleles with 55 total repeats.

Xpansion Interpreter requires three different PCR reactions for each DNA sample. Capillary electrophoresis (CE) of the products of one PCR reaction, called repeat primed PCR, examines this distribution to allow accurate determination of total repeat size (Figure 4A and reference 9). Characteristic signal “dips” occur at the site of AGG interruptions, as seen by the red arrows in Figure 4B and 4C.

Additional information is needed to determine the exact locations of the AGGs in females because the presence of two alleles creates interpretation challenges. Two additional PCR reactions allow resolution of the AGG position and number in females. Through specialized training to interpret the results from all three PCR reactions, the Asuragen CLIA laboratory staff that run Xpansion Interpreter are able to definitively determine the location of the AGG interruptions in both males and females.

Figure 4: Examples of the distribution of products from CGG repeat primed PCR. A) Uninterrupted male sample, B) Male sample with 3 AGG interruptions, C) Female sample with overlapping G interruptions. The red arrows point to where the AGG interruptions interfere with creation of PCR products.

In a blinded validation study using a set of whole blood specimens, Xpansion Interpreter revealed the comprehensive FMR1 genotype with 100% concordance to the reference method.

To allow clinical application of Xpansion Interpreter, Asuragen’s scientists collaborated with experts in the Fragile X community to conduct a study that determines the probability of expansion based on test results. Results of the study are summarized in Figure 1 and can be used as a tool to modify the probability that the allele will expand.

The following CPT codes will be used when billing for Xpansion Interpreter: 83891, 83892,83896, 83898, 83900, 83909, 83912

Results from Xpansion Interpreter testing can be expected within 7-10 business days after the patient's sample is received.