Ups and Downs: Mechanisms of Repeat Instability in the Fragile X-Related Disorders

Abstract

The Fragile X-related disorders (FXDs) are a group of clinical conditions resulting from the expansion of a CGG/CCG-repeat tract in exon 1 of the Fragile X mental retardation 1 (FMR1) gene. While expansions of the repeat tract predominate, contractions are also seen with the net result being that individuals can show extensive repeat length heterogeneity in different tissues. The mechanisms responsible for expansion and contraction are still not well understood. This review will discuss what is known about these processes and current evidence that supports a model in which expansion arises from the interaction of components of the base excision repair, mismatch repair and transcription coupled repair pathways.

Keywords: Fragile X syndrome; Fragile X-related disorders; base excision repair; mismatch repair; repeat expansion; transcription coupled repair.

Figure 1

Timing and tissue distribution of expansions in the Fragile X-related disorders (FXDs). The data for this figure reflects a synthesis of data from humans and a mouse model of the Fragile X premutation (FX PM). Intergenerational expansions likely occur prezygotically and perhaps in the very early embryo. Both maternal and paternal expansions are observed. However, as the repeat number increases so the frequency of paternally transmitted contractions increases [19]. In contrast, expansions that generate larger alleles predominate in females, with the likelihood that a PM allele will be transformed into a full mutation (FM) allele reaching 100% as the repeat number approaches 90 [19]. In humans, somatic expansions have been observed in blood and brain. Work in the FX PM mouse suggests that expansions can also occur in organs like liver and kidney, with more somatic expansions being observed in males than females. No contractions have been seen in the somatic tissue of adult animals. The extent of expansion and contraction is indicated by the font size of the associated text.

Figure 2

Generic representation of some of the unusual DNA structures described for the FX repeats. The arrows indicate single-stranded regions. Both DNA strands can form hairpins containing a mixture of Watson-Crick G•C base pairs (black dashed lines) and either G•G or C•C mispairs in the case of the CGG-strand and CCG-strand, respectively (gray dashed lines) [30,31,32,33,39]. Hairpin formation on one strand may facilitate hairpin formation on the other. This could result in the formation of Slipped DNA (S-DNA) [40] if the hairpins are offset. Quadruplexes may involve G4-tetrads and/or GCGC-tetrads (gray parallelograms) with C•C mismatches (dashed lines) [29,31,41,42,43]. The repeat tract can also form an R-loop containing an RNA:DNA hybrid formed between CGG-repeats in the transcript and the CCG-repeats in the DNA template [35,36,44]. In principle this hybrid could form in cis (perhaps co-transcriptionally) or in trans.

Figure 3

Types of repeat instability at the Fragile X Mental Retardation 1 (FMR1) locus. Evidence from a mouse FX PM model suggests that processing of the FX repeats during replication and repair can lead to either expansion, contraction or error-free repair. Expansion is by far the dominant mechanism with >80% of alleles showing expansion in male mice at 6 months of age [7]. About 2% of expansions involve the mismatch repair protein MutSα, with the remaining expansions involving either the mismatch repair protein MutSβ or a combination of MutSα and MutSβ [48,49]. DNA Polymerase β (Polβ), a polymerase essential for base excision repair, is also an important contributor to expansion with the Cockayne Syndrome Group B (CSB) protein, that is normally involved in Transcription Coupled Repair (TCR), playing an auxiliary role in older animals [50]. Contractions and error-free repair occur at about the same frequency, with error-free repair involving CSB [51], MutSα [49], as well as the DNA damage checkpoint proteins Ataxia Telangiectasia Mutated (ATM) and Ataxia Telangiectasia and Rad3-Related (ATR) [52,53]. Proteins involved in generating contractions have not yet been identified.

Figure 4

Model for repeat contraction and loss of AGG interruptions. During chromosomal replication or repair synthesis, strand-slippage within the repeat can result in a loop-out forming either on the nascent strand (not illustrated) or the template strand. A loop-out on the slipped nascent strand is thought to result in expansions by causing repriming to occur more 3′ on the template, while a loop-out on the template strand results in repriming more 5′ on the template leading to contractions. In a FX PM mouse model, a bimodal distribution of contraction sizes is seen with some alleles having lost 1–2 repeats and other having lost >7 [48]. Small contractions may result from the looping out of 1–2 repeats while larger contractions arise from the formation of larger loop-outs that may be stabilized by hydrogen bonding. If the template-strand loop-out contains the AGG interruption, then the interruption would be lost.