Mechanisms of Genome Instability in the Fragile X-Related Disorders

Abstract

The Fragile X-related disorders (FXDs), which include the intellectual disability fragile X syndrome (FXS), are disorders caused by expansion of a CGG-repeat tract in the 5' UTR of the X-linked FMR1 gene. These disorders are named for FRAXA, the folate-sensitive fragile site that localizes with the CGG-repeat in individuals with FXS. Two pathological FMR1 allele size classes are distinguished. Premutation (PM) alleles have 54-200 repeats and confer the risk of fragile X-associated tremor/ataxia syndrome (FXTAS) and fragile X-associated primary ovarian insufficiency (FXPOI). PM alleles are prone to both somatic and germline expansion, with female PM carriers being at risk of having a child with >200+ repeats. Inheritance of such full mutation (FM) alleles causes FXS. Contractions of PM and FM alleles can also occur. As a result, many carriers are mosaic for different sized alleles, with the clinical presentation depending on the proportions of these alleles in affected tissues. Furthermore, it has become apparent that the chromosomal fragility of FXS individuals reflects an underlying problem that can lead to chromosomal numerical and structural abnormalities. Thus, large numbers of CGG-repeats in the FMR1 gene predisposes individuals to multiple forms of genome instability. This review will discuss our current understanding of these processes.

Keywords: aneuploidy; base excision repair (BER); break induced replication (BIR); chromosome fragility; microhomology mediated end-joining (MMEJ); mitotic DNA synthesis (MiDAS); repeat contractions; repeat expansion; repeat mosaicism.

Figure 1

Model for mitotic DNA synthesis (MiDAS) at the FMR1 locus. FM alleles with stalled replication forks that have not been resolved by the time mitosis begins are cleaved by the SLX1/SLX4 nuclease to generate a one-ended DSB. After end resection to generate a 3′ overhang, binding of RAD51 allows the exposed 3′ end to invade a homologous template, likely a sister chromatid. Conservative DNA synthesis then occurs in a POLD3-dependent fashion to complete replication [48]. Failure to initiate this process results in the formation of ultrafine bridges and the high frequency loss of the affected X chromosome [26,48], while failure to complete MiDAS results in fragile site expression [48].

Figure 2

Diagrammatic representation of possible transcription-related, MMR-dependent events that could give rise to repeat expansions. As illustrated on the left-hand side of this figure, R-loops could give rise to expansions because single stranded regions of the R-loop would be prone to oxidative damage. Repair of this damage by BER would create an opportunity for strand slippage and strand-displacement that could result in the formation of hairpins or loop-outs on one or both strands [83]. Alternatively, as illustrated on the right, hairpin formation might occur on the non-template strand of the R-loop, forming an S-loop. This might favor formation of a hairpin on the template strand after dissociation of the transcript. Hairpins or double loop-outs formed by either process could then be bound by both MutS proteins. This results in the recruitment of MutLα, MutLβ, and MutLγ. MutLγ cleavage of the strands opposite each loop-out would generate a DSB that would then be repaired by some, as yet unknown, non-homologous end-joining-independent DSBR pathway.

Figure 3

Diagrammatic representation of potential contraction/deletion pathways. (A) Strand-slippage during replication may be exacerbated by hairpins formed on the template strand. Repriming more 5′ on the template would lead to nascent strands with fewer repeats than the template strand. A subsequent round of replication would generate a contracted allele. (B) MMEJ-mediated repair of a DSB is initiated by end-resection to reveal MHs at either side of the break. Annealing of these MHs is followed by removal of non-homologous flaps, filling of any gaps, perhaps by Polθ or Polβ, and ligation by either ligase 3 (Lig3) or ligase 1 (Lig1).

Figure 4

Summary of potential sources of genomic instability associated with PM and FM alleles. Transcription of long CGG-repeat tracts promotes a form of MMR-dependent DSBR that leads to expansion (i). As the repeat number increases, the incidence of replication fork stalling increases. Repair of the stalled fork can lead to contractions or deletions (ii). Aneuploidy can result when stalled forks are not repaired (iii), while chromosome fragility results when repair of the stalled forks by MiDAS is incomplete (iv).