Screening for CGG Repeat Expansion in the FMR1 Gene by Melting Curve Analysis of Combined 5′ and 3′ Direct Triplet-Primed PCRs

Abstract

CGG repeat expansions in the FMR1 (fragile X mental retardation 1) gene are associated with fragile X syndrome, fragile X-associated tremor/ataxia syndrome, and fragile X-associated primary ovarian insufficiency. We evaluated the use of melting curve analysis (MCA) of triplet-primed PCR (TP-PCR) assays as a rapid screening tool for the positive identification of expanded FMR1 alleles in men and women. Both 5'- and 3'-weighted direct TP-PCRs (dTP-PCRs) were evaluated on 29 cell line-derived DNA samples and 44 blinded clinical samples. The presence of expansions was identified by the melting curve profiles generated automatically through MCA on the LightCycler 480 Real-Time PCR System. All samples were also analyzed by capillary electrophoresis to confirm the identities of the PCR fragments that gave rise to the observed melt peak profiles. The presence of expanded alleles in samples from both males and females produced melt peak profiles that were distinct from those of individuals with the normal allelic form. In the blinded test, positive and negative calls for the presence of an expanded allele corroborated with previously determined genotype classifications for all samples. The approach of dTP-PCR plus MCA offers a single-step strategy with high diagnostic sensitivity and specificity for rapid screening detection of FMR1 CGG repeat expansions, regardless of sex. The combined use of 5'- and 3'-weighted dTP-PCR assays minimizes the incidence of false-negative results arising from repeat-flanking deletions.