2012-06-20 10.00 Reclassification of FMR1 expansion risk The role of AGG interruptions from Asuragen on Vimeo.

Dr. Liz Berry-Kravis Power Point Slides
  • Presented by Dr. Liz Berry-Kravis, Director of Molecular Laboratory
    Rush-Presbyterian, St Lukes’ Medical Center

Fragile X Facts

The average age when FXS is diagnosed is about 36 months for boys, and 42 months for girls. It has been reported that up to 40% of families with FXS girls, and 25% with FXS boys, have had another child before their first child was diagnosed.
Since they have an extra X chromosome that is usually normal, females with FXS are usually less severely affected than males.

AGG Interruptions - Fragile X

In normal individuals, the triplet repeat segment is not comprised exclusively of consecutive CGG repeats. Instead, normal alleles commonly contain blocks of these repeats that are interspersed with AGG sequences. These “AGG interruptions” usually occur with a periodicity of 9–10 CGG repeat units, with most alleles containing a total of 1-3 AGG sequences. The frequency and position of interrupting AGG units has been associated with the stability of the repeat. Several published, but preliminary, studies have suggested that AGG sequences interspersed along CGG repeat tract in FMR1 carriers can influence the expansion of the triplet repeat tract in the next generation [1,2,3]. For example, Eichler et al [1] reported that a threshold of 34-38 uninterrupted CGG repeats was associated with gene instability in a study of 28 parent-to-child transmissions. However, until recently, methods to detect AGG interruptions were cumbersome, low throughput, and difficult to interpret, particularly in female samples. As a result, the risk of FMR1 expansion in the next generation is currently estimated from the total number of CGG repeats in the 5′ untranslated region [3] rather than from a comprehensive genotype that includes both the CGG and AGG structure.

The lack of a definitive, routine technology for mapping FMR1 AGG interruptions has prevented a large, multicenter study of the impact of the comprehensive genotype on the risk of expansion. The recent development and optimization of a set of efficient, long-read PCR reagents [4,5] however, combined with new interpretive tools [5] has now surmounted this limitation. As a result, the distinct AGG structure of even complex samples with multiple alleles can be readily resolved.[5]

Webinar Objectives:

  • • To understand the relationship between AGG interruptions, consecutive CGG repeats, and FMR1 transmission stability
  • • Understand the application of new technology in assessing risk to expansion in females with intermediate or premutation alleles
  • • Discuss the counseling implications of more informative risk assessments that consider AGG status
  • • Review data from a collaborative study of >500 mother-to-child transmissions of alleles


1. Length of uninterrupted CGG repeats determines instability in the FMR1 gene.
Eichler EE et al. Nat Genet 1994; 8(1):88-94
**This is the primary paper identifying AGG as a stabilizing factor in preventing expansion of the CGG repeat region. Out of 84 chromosomes analyzed for AGG, all documented unstable transmissions were due to a loss of one or more AGG defining an instability threshold of 34-38 uninterrupted CGG repeats. The authors suggest that loss of AGG is an important mutational event in the generation of unstable alleles predisposed to the fragile X syndrome.

2. Fragile X "gray zone" alleles: AGG patterns, expansion risks, and associated haplotypes.
Zhong N et al. Cell 1994; 77(6):853-61;

3. Expansion of the fragile X CGG repeat in females with premutation or intermediate alleles.
Nolin SL et al. Am J Hum Genet 2003; 72(2):454-464

4. A Novel FMR1 PCR method for the routine detection of low abundance expanded alleles and full mutations in Fragile X Syndrome.
Filipovic-Sadic S et al. Clin Chem 2010; 56(3): 399-408

5. An information-rich CGG repeat primed PCR that detects the full range of Fragile X expanded alleles and minimizes the need for Southern blot analysis.
Chen, L et al. J Mol Diag 2010; 12(5): 589-600
**The authors describe a PCR method suitable for the detection and sizing of normal, intermediate, premutation and full mutation alleles in which a consequential outcome was identification of the number and location of interrupting sequences. The authors report resolution in male samples and inferred resolution in female samples relative to DNA sequencing analysis.

6. Cryptic and polar variation of the fragile X repeat could result in predisposing normal alleles.
Kunst CB et al. Cell 1994; 77(6):853-861

7. Fragile X Gene Instability: Anchoring AGGs and Linked Microsatellites.
Zhong N et al. Am J Hum Genet 1995; 57(2): 351-361
*The authors expand on the analysis of AGG repeats using partial restriction digestion and fragment sizing to determine AGG status in 187 males affirming association of the presence of AGG with stability of the repeat region.

8. Familial transmission of the FMR1 CGG repeat.
Nolin SL et al. Am J Hum Genet 1996; 59(6):1252-1261

9. Examination of Factors Associated with Instability of the FMR1 CGG Repeat.
Ashley-Koch AE et al. Am J Hum Genet 1998; 63(3): 776-785

10. Fragile X CGG repeat structures among African-Americans: identification of a novel factor responsible for repeat instability.
Crawford DC et al. Hum Mol Genet 2000; 9(12): 1759-1769

11. AGG interspersion analysis of the FMR1 CGG repeats in mental retardation of unspecific cause.
Poon PM et al. Clin Biochem 2006; 39(3): 244-248

12. Analysis of the Fragile X Trinucleotide Repeat in Basques: Association of Premutation and Intermediate Sizes, Anchoring AGGs and Linked Microsatellites with Unstable Alleles.
Arrieta MI et al. Curr Genomics 2008; 9(3): 191-199

13. Expansion of an FMR1 grey-zone allele to a full mutation in two generations.
Fernandez-Carvajal I et al. J Mol Diagn 2009; 11(4):306-310
**This paper tracks the expansion of a 53 CGG repeat in a grandfather with 2 AGG to a 56 CGG repeat in his daughter (0 AGG) and subsequent full mutation allele in the grandson. This familial study documents the lowest allele size known to expand to a full repeat and the importance of the associated AGG status.

14. The role of AGG interruptions in the transcription of FMR1 premutation alleles.
Yrigollen CM at al. PLoS One 2011; 6(7):e21728. Epub 2011 Jul 19

15. Fragile X analysis of 1112 prenatal samples from 1991 to 2010.
Nolin SL et al. Prenat Diagn 2011; 31(10):925-931
**The authors present results of allele transmissions in over 1000 samples helping to segregate risk factors for expansion and to improve genetic counseling of women with and without a family history of fragile X syndrome. Within this group, however, there will be a few women at high risk for large expansions although these cannot be differentiated at the present time from others with a low risk. Broader studies on the influence of AGG status on risk for expansion are suggested.

This is an educational website sponsored by Asuragen and not funded by or hosted for any advertising source