Mechanism Of Repeat Expansion Research Articles

Mystery of Expansion: DNA Metabolism and Unstable Repeats.

The mammalian genome mostly contains repeated sequences. Some of these repeats are in the regulatory elements of genes, and their instability, particularly the propensity to change the repeat unit number, is responsible for 36 well-known neurodegenerative human disorders. The mechanism of repeat expansion has been an unsolved question for more than 20years. There are a few hypotheses describing models of mutation development. Every hypothesis is based on assumptions about unusual secondary structures that violate DNA metabolism processes in the cell. Some models are based on replication errors, and other models are based on mismatch repair or base excision repair errors. Additionally, it has been shown that epigenetic regulation of gene expression can influence the probability and frequency of expansion. In this review, we consider the molecular bases of repeat expansion disorders and discuss possible mechanisms of repeat expansion during cell metabolism.

DNA repair/replication transcripts are down regulated in patients with Fragile X Syndrome

BackgroundFragile X Syndrome (FXS) and its associated disorders are caused by the expansion of the CGG repeat in the 5’ untranslated region of the fragile X mental retardation 1 (FMR1) gene, with disease classification based on the number of CGG repeats. The mechanisms of repeat expansion are dependent on the presence of cis elements and the absence of trans factors both of which are not mutually exclusive and contribute to repeat instability. Expansions associated with trans factors are due to the haploinsuffient or reduced expression of several DNA repair/metabolizing proteins. The reduction of expression in trans factors has been primarily conducted in animal models without substantial examination of many of these expansion mechanisms and trans factors in humans.ResultsTo understand the trans factors and pathways associated with trinucleotide repeat expansion we have analyzed two microarray datasets which characterized the transcript expression in patients with FXS and in controls.ConclusionWe observed significant down regulation of DNA damage/repair pathway transcripts. This observation was consistent in both datasets, which used different populations. Within these datasets, several transcripts overlapped in the direction of association and fold change. Further characterization of these genes will be critical to understand their role in trinucleotide repeat instability in FXS.

No association of the SCA1 (CAG)31 allele with Huntington's disease, myotonic dystrophy type 1 and spinocerebellar ataxia type 3.

Trinucleotide repeat expansions are the underlying mutation in several neurodegenerative and neuromuscular disorders including at least eight spinocerebellar ataxias (SCA). The molecular mechanisms of repeat expansion are as yet insufficiently understood. Recently, an association of the SCA1 (CAG)31 repeat allele with Huntington's disease and myotonic dystrophy type 1 was described. These findings implicate a possible role of the SCA1 (CAG)31 allele in other triplet diseases. We analyzed the SCA1 CAG repeat length in a large sample of Huntington's disease (n=182), myotonic dystrophy type 1 (n=64) and SCA3 (n=31) patients. In none of these groups was a significant association with the 31 repeat allele found. Our findings do not support the hypothesis that this allele is involved in the etiology of trinucleotide expansion.

Instability of a (CGG)98 repeat in the Fmr1 promoter.

Fragile X syndrome is one of 14 trinucleotide repeat diseases. It arises due to expansion of a CGG repeat which is present in the 5'-untranslated region of the FMR1 gene, disruption of which leads to mental retardation. The mechanisms involved in trinucleotide repeat expansion are poorly understood and to date, transgenic mouse models containing transgenic expanded CGG repeats have failed to reproduce the instability seen in humans. As both cis-acting factors and the genomic context of the CGG repeat are thought to play a role in expansion, we have now generated a knock-in mouse Fmr1 gene in which the murine (CGG)8 repeat has been exchanged with a human (CGG)98 repeat. Unlike other CGG transgenic models, this model shows moderate CGG repeat instability upon both in maternal and paternal transmission. This model will now enable us to study the timing and the mechanism of repeat expansion in mice.

Apparent FMR1 allele instability in non-fragile X males.

241 TZOUNTZOURIS et al. reported on apparent FMR1 allele instability of 9 developmentally delayed individuals with normal-sized alleles. This raises interesting questions and possible concerns regarding laboratory genetic testing of mutated Fragile X genes. The FMR1 CGG repeat is polymorphic ( , 10–55 repeats) in the normal population, with 30 repeats being the most common size (Brown et al., 1993). These normal alleles are generally considered to be very stable. However, their findings suggest that even normal sized alleles may, on occasion, have instability. Their documentation of this observation suggests laboratories doing such testing should be aware of the potential for this confusing finding to appear in their samples. Analysis of the repeat in normal males reveals AGG triplets embedded at regular intervals, most commonly at repeat positions of about 10 and 20 (Eichler et al., 1994; Kunst et al., 1994; Zhong et al., 1995). The alleles in Fragile X families differ in several ways. They have long repeats that are unstably transmitted from one generation to the next. Repeat expansions are gender-specific, with expansion to full-mutation alleles occurring only through females. In premutation females, the expansions are a function of repeat size, with larger repeats having a greater risk of expansion than smaller ones (Nolin et al., 1996, 1999). Analysis of premutation males indicates that Fragile X alleles usually have one or no AGG interruptions within the repeat and long stretches of pure CGG repeats in the 39 end. Despite intense interest in trinucleotide disorders, the mechanism of repeat expansion is poorly understood. For Fragile X, the expansion process is especially puzzling because the repeat may expand, for example, from 75 to more than 1,000 in one generation. The presence of long, uninterrupted CGG tracts in Fragile X families clearly distinguishes them from other families. The current favored model for large repeat expansions is slippage of Okazaki fragments during DNA replication of repeat regions (Brown et al., 1993; Richards and Sutherland, 1994). Expansions in Fragile X and the other trinucleotide disorders are not likely to be the result of defective DNA replication/repair proteins because the instability is limited to a single expanded repeat, and global microsatellite instability is not observed. Recent studies suggest that repeat instability is a consequence of abnormal localized DNA secondary structure (McMurray, 1999). Biochemical studies have demonstrated the ability of CGG tracts to form alternative DNA structures. Strand displacement of the repeat regions in Okazaki fragments may allow the formation of intramolecular hairpin and tetrahelical structures. These structures may defeat the DNA replication/repair machinery within a cell. Several DNA repair-related enzymes have been recently implicated in trinucleotide repeat instability. The FEN-1 enzyme is both an endonuclease and exonuclease involved in DNA replication and repair. One preferred substrate is branched DNA structures with a single-stranded 59 flap, such as Okazaki fragments generated from the lagging strand. In contrast, secondary structures such as loops and hairpins are resistant to cleavage by FEN-1. Impaired FEN-1 function due to inefficient cleavage of secondary structures in Okazaki fragments with long stretches of triplet repeats may lead to repeat expansion, whereas structures formed in the lagging strand during replication of CGG repeats can cause replication stalling (Goredin et al., 1997). The WRN gene is a DNA helicase responsible for Werner syndrome, an autosomal recessive disease characterized as a premature aging condition. It appears to be involved in DNA replication, both as a helicase and as a 39 to 59 exonuclease. The WRN helicase has been shown to unwind CGG/AGG oligomers more rapidly than uninterrupted CGG oligomers, suggesting that it is involved in repairing abnormal DNA structures such as DNA tetrahelices (Weisman-Shomer et al., 2000). Fragile X syndrome is a complex disorder that is poorly understood. Although the mutation is known to be an expansion of a CGG repeat, the factors leading to repeat expansion remain obscure. The findings in the current report (Tzountzouris et al., this issue) raise interesting questions regarding the mechanisms of normal allele length instability and imply that laboratories need to be on the look-out for their estimated 1% of cases that can lead to diagnostic confusion.