Recent advances in assays for the fragile X-related disorders

Abstract

The fragile X-related disorders are a group of three clinical conditions resulting from the instability of a CGG-repeat tract at the 5' end of the FMR1 transcript. Fragile X-associated tremor/ataxia syndrome (FXTAS) and fragile X-associated primary ovarian insufficiency (FXPOI) are disorders seen in carriers of FMR1 alleles with 55-200 repeats. Female carriers of these premutation (PM) alleles are also at risk of having a child who has an FMR1 allele with >200 repeats. Most of these full mutation (FM) alleles are epigenetically silenced resulting in a deficit of the FMR1 gene product, FMRP. This results in fragile X Syndrome (FXS), the most common heritable cause of intellectual disability and autism. The diagnosis and study of these disorders is challenging, in part because the detection of alleles with large repeat numbers has, until recently, been either time-consuming or unreliable. This problem is compounded by the mosaicism for repeat length and/or DNA methylation that is frequently seen in PM and FM carriers. Furthermore, since AGG interruptions in the repeat tract affect the risk that a FM allele will be maternally transmitted, the ability to accurately detect these interruptions in female PM carriers is an additional challenge that must be met. This review will discuss some of the pros and cons of some recently described assays for these disorders, including those that detect FMRP levels directly, as well as emerging technologies that promise to improve the diagnosis of these conditions and to be useful in both basic and translational research settings.

Fig. 1. Southern blotting assay for the FMR1-related disorders

A) Diagrammatic representation of the 5.2 kb EcoRI fragment containing the 5’ end of the FMR1 gene. The coordinates are from the GRCh38/hg38 build of the human genome. The location of various commonly used methylation-sensitive restriction enzyme recognition sites, as well as the location of the CGG repeat tract and the 2 sets of commonly used probes are shown. The predicted fragment sizes that would be obtained from an unmethylated and a methylated allele is illustrated below. B) A graphic representation of the expected fragment sizes that would be seen in male and female carriers of normal, PM and FM alleles including carriers of unmethylated FM (UFM) alleles and a male mosaic for both a methylated FM allele and a heterogenous mixture of unmethylated PM and FM alleles. In practice samples can be more heterogeneous and the signal to noise ratio lower than depicted here.

Fig. 2. Schematic representation of the various amplification-based assays for the extent of methylation in the FMR1-related disorders

The asterisk indicates an assay that is partially quantitative in being able to distinguish between normal males and males with and without methylation mosaicism. However, it is not sensitive enough for use in females. Allele-specific methylation refers to the ability of the assay to identify which allele is methylated, knowledge that is relevant in females where skewed XCI may modify disease severity. The MLPA assay can detect abnormal FMR2 methylation and some contractions. In principle, the probe set could be modified to include those for other genetic disorders. The methylation array assay can detect abnormal methylation genome-wide and can thus also detect imprinting disorders.

Fig. 3. Triplet-primed assays for the detection of AGG interruptions

A–B) Potential primer binding sites in the first generation (CGG-primed) and second generation (A-primed) reactions. The grey parallelograms in panel A illustrate the places on the template that the primer will not bind. The red asterisk indicates the fluorescently labeled primer. C) The output of the CGG-primed reaction on DNA from a female PM carrier. The open arrowhead in panel C marks the PCR product resulting from the flanking PCR primers on the normal allele, while the grey arrowhead indicates the PCR product resulting from the use of the flanking primers on the PM allele. The arrows indicate the PCR products resulting from the use of the triplet containing primer and the fluorescently labeled reverse primer. The inset is a magnification of the 400–700 bp region of the electrophoretogram. D) The output of the A-primed reaction on the same PM carrier shown in panel C. The open circles indicate the PCR products corresponding to priming at each of the 2 AGG interruptions in the normal allele while the filled circles indicate the PCR products corresponding to priming at each of the 2 AGG interruptions in the PMl allele. This figure was adapted from our previously published work for which we retain the copyright (Hayward and Usdin In Press).

Fig. 4. Ilustration of the principle of recently described dual-FMRP antibody assays for FMRP

The colors of the fluorescent/luminscent labels indicates the part of the electromagnetic spectrum in which the label emits light. The HTRF®-based FMRP assays involve a FMRP antibody coupled to either Europium cryptate (Kumari et al. 2015) as illustrated here, or Lumi4-Tb cryptate (Schutzius et al. 2013), and a second antibody tagged with an energy acceptor such as d2. Irradiation of the sample at ~337 nm excites the cryptate moiety and if the two antibodies bind to epitopes on FMRP that are within ~70–90 Å, FRET occurs. This results in the excitation of the second fluorophore and emission at 665 nm. The ELISA assay described for FMRP uses a chicken anti-FMRP antibody shown in blue to coat the well of the microplate and capture FMRP. The second FMRP antibody, shown in aqua, is a mouse monoclonal antibody used to detect FMRP via its interaction with a third antibody, shown in red, an anti-mouse IgG antibody conjugated to horseradish peroxidase (HRP). After addition of the HRP substrate, PS-atto, the samples are detected at 450 nm. The Luminex®-based assay for FMRP uses a mouse monoclonal FMRP antibody, shown in red, that is coupled to xMAP beads or microspheres to capture the FMRP. The second FMRP antibody, shown in purple, is a rabbit polyclonal Ab. Detection is carried out at 575 nm using a goat-anti-rabbit phycoerythrin-conjugated IgG antibody, shown in green. Note that while both the HTRF® and the Luminex®-based assays can in principle be multiplexed, the multiplexing potential of the Luminex®-based assay is much higher than that of HTRF® assays as denoted by the larger number of checkmarks.