Abstract
The learning disability fragile X syndrome results from the presence of >200 CGG/CCG repeats in exon 1 of the X-linked gene FMR1. Such alleles arise by expansion from maternally transmitted FMR1 premutation alleles, alleles having 55 to 200 repeats. Expansion risk is directly related to maternal repeat number. However, AGG interruptions to the repeat tract are important modifiers of expansion risk. Thus, the ability to identify such interruptions is crucial for the appropriate genetic counseling of females who are premutation carriers. First-generation triplet-primed PCR assays allow these interruptions to be detected. However, because the triplet primer used has multiple binding sites in the repeat tract, interpreting the results is not straightforward and it is not always possible to unambiguously determine the AGG-interruption status in females because of the difficulties associated with the presence of a second X chromosome. Interpretation is further complicated by any repeat size mosaicism that may be present. We have developed second-generation PCR assays that prime specifically at the interruptions. These assays are simpler to interpret and better able to evaluate this important determinant of expansion risk in females even in those with a mixture of premutation allele sizes.