Molecular Correlates and Recent Advancements in the Diagnosis and Screening of FMR1-Related Disorders

Abstract

Fragile X syndrome (FXS) is the most common monogenic cause of intellectual disability and autism. Molecular diagnostic testing of FXS and related disorders (fragile X-associated primary ovarian insufficiency (FXPOI) and fragile X-associated tremor/ataxia syndrome (FXTAS)) relies on a combination of polymerase chain reaction (PCR) and Southern blot (SB) for the fragile X mental retardation 1 (FMR1) CGG-repeat expansion and methylation analyses. Recent advancements in PCR-based technologies have enabled the characterization of the complete spectrum of CGG-repeat mutation, with or without methylation assessment, and, as a result, have reduced our reliance on the labor- and time-intensive SB, which is the gold standard FXS diagnostic test. The newer and more robust triplet-primed PCR or TP-PCR assays allow the mapping of AGG interruptions and enable the predictive analysis of the risks of unstable CGG expansion during mother-to-child transmission. In this review, we have summarized the correlation between several molecular elements, including CGG-repeat size, methylation, mosaicism and skewed X-chromosome inactivation, and the extent of clinical involvement in patients with FMR1-related disorders, and reviewed key developments in PCR-based methodologies for the molecular diagnosis of FXS, FXTAS and FXPOI, and large-scale (CGG)_n expansion screening in newborns, women of reproductive age and high-risk populations.

Keywords: AGG interruption; CGG repeat; FMR1; FXPOI; FXTAS; fragile X syndrome; melting curve analysis; methylation; screening; triplet-primed PCR.

Figure 1

Flanking or repeat-spanning PCR: (A) Schematic depicting the flanking PCR primer design; and (B) expected capillary electrophoresis profiles of normal, premutation and full-mutation males and females.

Figure 2

Triplet-Primed PCR or TP-PCR: (A) Schematic depicting the TP-PCR primer design; and (B) expected capillary electrophoresis profiles of normal, premutation and full-mutation males and females. For simplification, the Tail primer is not shown.

Figure 3

Melting curve analysis (MCA) of TP-PCR amplicons. Schematic illustrating how MCA of heterogeneous TP-PCR amplicons results in the generation of a single melt peak. Dissociation of double-stranded PCR products and re-distribution of SYBR Green I Dye from the shorter to larger fragments with gradually increasing temperature (Temp A to Temp C) is shown. At Temp D, when the largest amplicon strands dissociate, SYBR Green I is completely released and remains unbound to DNA, resulting in a sharp decrease in fluorescence. −dF/dT, first negative derivative of fluorescence versus temperature.