About Xpansion Interpreter®
AGG interruptions within the CGG repeats of the FMR1 gene have long been suggested to stabilize alleles to protect against expansion, yet to date, genetic counseling relies upon the total number of CGG repeats. In fact, the number of 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 1,040 parent-to-child transmissions for alleles with 45-90 total repeats (Nolin, et al. 2015 and Nolin, et al. 2013). The studies showed a clear modification of the probability of expansion, of variable sizes and degrees, when number and location of AGG interruptions and number of CGG repeats were considered.
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 2A). Characteristic signal “dips” occur at the site of AGG interruptions, as seen by the red arrows in Figure 2B and 2C. 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 2: 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 AGG 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 studies that determined the probability of expansion based on test results. Results of the studies can be used as a tool to modify the probability that the allele will expand to a fragile X full mutation.
Case 1: Smallest reported expansion to full mutation.
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 (Fernandez-Carvajal I et al. 2009). 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.
Impact of AGG Interruptions:
The likelihood for expansion to a fragile X full mutation is based on the total number of CGG repeats and the number of AGG interruptions (1,2). In the figure below, the grey bars represent the risk of expansion to a full mutation in the absence of knowledge of number of AGG interruptions. The lines represent the modified risk when AGG information is incorporated (1,2). As an example, an FMR1 allele with 75-79 CGG repeats has a 10-fold greater risk of expansion with 0 AGG interruptions (74%) compared to 2 AGG interruptions (7%).
1. Nolin SL et al. Fragile X AGG analysis provides new risk predictions for 45-69 repeat alleles. Am J Med Genet 2013; 161(4):771-8.
2. Nolin SL et al. Fragile X full mutation expansions are inhibited by one or more AGG interruptions in premutation carriers. Genet Med 2015 May;17(5):358-64.