Structural basis for triplet repeat disorders: a computational analysis

MOTIVATION: Over a dozen major degenerative disorders, including myotonic distrophy, Huntington's disease and fragile X syndrome, result from unstable expansions of particular trinucleotides. Remarkably, only some of all the possible triplets, namely CAG/CTG, CGG/CCG and GAA/TTC, have been associated with the known pathological expansions. This raises some basic questions at the DNA level. Why do particular triplets seem to be singled out? What is the mechanism for their expansion and how does it depend on the triplet itself? Could other triplets or longer repeats be involved in other diseases? RESULTS: Using several different computational models of DNA structure, we show that the triplets involved in the pathological repeats generally fall into extreme classes. Thus, CAG/CTG repeats are particularly flexible, whereas GCC, CGG and GAA repeats appear to display both flexible and rigid (but curved) characteristics depending on the method of analysis. The fact that (1) trinucleotide repeats often become increasingly unstable when they exceed a length of approximately 50 repeats, and (2) repeated 12-mers display a similar increase in instability above 13 repeats, together suggest that approximately 150 bp is a general threshold length for repeat instability. Since this is about the length of DNA wrapped up in a single nucleosome core particle, we speculate that chromatin structure may play an important role in the expansion mechanism. We furthermore suggest that expansion of a dodecamer repeat, which we predict to have very high flexibility, may play a role in the pathogenesis of the neurodegenerative disorder multiple system atrophy (MSA). CONTACT: pfbaldi@ics.uci.edu, yves@netid.com, brunak@cbs.dtu.dk, gorm@cbs.dtu.dk.     

A SCA7 CAG/CTG repeat expansion is stable in Drosophila melanogaster despite modulation of genomic context and gene dosage

CAG and CTG repeat expansions are the cause of at least a dozen inherited neurological disorders. In these so-called "dynamic mutation" diseases, the expanded repeats display dramatic genetic instability, changing in size when transmitted through the germline and within somatic tissues. As the molecular basis of the repeat instability process remains poorly understood, modeling of repeat instability in model organisms has provided some insights into potentially involved factors, implicating especially replication and repair pathways. Studies in mice have also shown that the genomic context of the repeat sequence is required for CAG/CTG repeat instability in the case of spinocerebellar ataxia type 7 (SCA7), one of the most unstable of all CAG/CTG repeat disease loci. While most studies of repeat instability have taken a candidate gene approach, unbiased screens for factors involved in trinucleotide repeat instability have been lacking. We therefore attempted to use Drosophila melanogaster to model expanded CAG repeat instability by creating transgenic flies carrying trinucleotide repeat expansions, deriving flies with SCA7 CAG90 repeats in cDNA and genomic context. We found that SCA7 CAG90 repeats are stable in Drosophila, regardless of context. To screen for genes whose reduced function might destabilize expanded CAG repeat tracts in Drosophila, we crossed the SCA7 CAG90 repeat flies with various deficiency stocks, including lines lacking genes encoding the orthologues of flap endonuclease-1, PCNA, and MutS. In all cases, perfect repeat stability was preserved, suggesting that Drosophila may not be a suitable system for determining the molecular basis of SCA7 CAG repeat instability.     

Identification of an HD patient with a (CAG)180 repeat expansion and the propagation of highly expanded CAG repeats in lambda phage

The Huntington’s disease mutation has been identified as a CAG/polyglutamine repeat expansion in a large gene of unknown function. In order to develop the transgenic systems necessary to uncover the molecular pathology of this disorder, it is necessary to be able to manipulate highly expanded CAG repeats in a cloned form. We have identified a patient with an expanded allele of greater than 170 repeat units and have cloned the mutant allele in the lambda zap vector. The recovery of highly expanded repeats after clone propagation was more efficient when repeats were maintained as lambda phage clones rather than as the plasmid counterparts. Manipulation of the repeats as phage clones has enabled us to generate Huntington’s disease transgenic mice that contain highly expanded (CAG)₁₁₅–(CAG)₁₅₀ repeats and that develop a progressive neurological phenotype. Received: 7 October 1996 / Revised: 5 December 1996     

No evidence of expansion of CAG or GAA repeats in schizophrenia families and monozygotic twins

Many diseases caused by trinucleotide expansion exhibit increased severity and decreased age of onset (genetic anticipation) in successive generations. Apparent evidence of genetic anticipation in schizophrenia has led to a search for trinucleotide repeat expansions. We have used several techniques, including Southern blot hybridization, repeat expansion detection (RED) and locus-specific PCR to search for expanded CAG/CTG repeats in 12 families from the United Kingdom and 11 from Iceland that are multiplex for schizophrenia and demonstrate anticipation. The unstable DNA theory could also explain discordance of phenotype for schizophrenia in pairs of monozygotic twins, where the affected twin has a greater number of repeats than the unaffected twin. We used these techniques to look for evidence of different CAG/CTG repeat size in 27 pairs of monozygotic twins who are either concordant or discordant for schizophrenia. We have found no evidence of an increase in CAG/CTG repeat size for affected members in the families, or for the affected twins in the MZ twin sample. Southern hybridization and RED analysis were also performed for the twin and family samples to look for evidence of expansion of GAA/TTC repeats. However, no evidence of expansion was found in either sample. Whilst these results suggest that these repeats are not involved in the etiology of schizophrenia, the techniques used for detecting repeat expansions have limits to their sensitivity. The involvement of other trinucleotide repeats or other expandable repeat sequences cannot be ruled out. Received: 8 September 1997 / Accepted: 13 March 1998     

Highly Specific Contractions of a Single CAG/CTG Trinucleotide Repeat by TALEN in Yeast

Trinucleotide repeat expansions are responsible for more than two dozens severe neurological disorders in humans. A double-strand break between two short CAG/CTG trinucleotide repeats was formerly shown to induce a high frequency of repeat contractions in yeast. Here, using a dedicated TALEN, we show that induction of a double-strand break into a CAG/CTG trinucleotide repeat in heterozygous yeast diploid cells results in gene conversion of the repeat tract with near 100% efficacy, deleting the repeat tract. Induction of the same TALEN in homozygous yeast diploids leads to contractions of both repeats to a final length of 3–13 triplets, with 100% efficacy in cells that survived the double-strand breaks. Whole-genome sequencing of surviving yeast cells shows that the TALEN does not increase mutation rate. No other CAG/CTG repeat of the yeast genome showed any length alteration or mutation. No large genomic rearrangement such as aneuploidy, segmental duplication or translocation was detected. It is the first demonstration that induction of a TALEN in an eukaryotic cell leads to shortening of trinucleotide repeat tracts to lengths below pathological thresholds in humans, with 100% efficacy and very high specificity.     

Mutation detection in Machado-Joseph disease using repeat expansion detection.

BACKGROUND: Several neurological disorders have recently been explained through the discovery of expanded DNA repeat sequences. Among these is Machado-Joseph disease, one of the most common spinocerebellar ataxias (MJD/SCA3), caused by a CAG repeat expansion on chromosome 14. A useful way of detecting repeat sequence mutations is offered by the repeat expansion detection method (RED), in which a thermostable ligase is used to detect repeat expansions directly from genomic DNA. We have used RED to detect CAG expansions in families with either MJD/SCA3 or with previously uncharacterized spinocerebellar ataxia (SCA). MATERIALS AND METHODS: Five MJD/SCA3 families and one SCA family where linkage to SCA1-5 had been excluded were analyzed by RED and polymerase chain reaction (PCR). RESULTS: An expansion represented by RED products of 180-270 bp segregated with MJD/SCA3 (p < 0.00001) in five families (n = 60) and PCR products corresponding to 66-80 repeat copies were observed in all affected individuals. We also detected a 210-bp RED product segregating with disease (p < 0.01) in a non-SCA1-5 family (n = 16), suggesting involvement of a CAG expansion in the pathophysiology. PCR analysis subsequently revealed an elongated MJD/SCA3 allele in all affected family members. CONCLUSIONS: RED products detected in Machado-Joseph disease families correlated with elongated PCR products at the MJD/SCA3 locus. We demonstrate the added usefulness of RED in detecting repeat expansions in disorders where linkage is complicated by phenotyping problems in gradually developing adult-onset disorders, as in the non-SCA1-5 family examined. The RED method is informative without any knowledge of flanking sequences. This is particularly useful when studying diseases where the mutated gene is unknown. We conclude that RED is a reliable method for analyzing expanded repeat sequences in the genome.     

Replication and expansion of trinucleotide repeats in yeast

The mechanisms of trinucleotide repeat expansions, underlying more than a dozen hereditary neurological disorders, are yet to be understood. Here we looked at the replication of (CGG)(n) x (CCG)(n) and (CAG)(n) x (CTG)(n) repeats and their propensity to expand in Saccharomyces cerevisiae. Using electrophoretic analysis of replication intermediates, we found that (CGG)(n) x (CCG)(n) repeats significantly attenuate replication fork progression. Replication inhibition for this sequence becomes evident at as few as approximately 10 repeats and reaches a maximal level at 30 to 40 repeats. This is the first direct demonstration of replication attenuation by a triplet repeat in a eukaryotic system in vivo. For (CAG)(n) x (CTG)(n) repeats, on the contrary, there is only a marginal replication inhibition even at 80 repeats. The propensity of trinucleotide repeats to expand was evaluated in a parallel genetic study. In wild-type cells, expansions of (CGG)(25) x (CCG)(25) and (CAG)(25) x (CTG)(25) repeat tracts occurred with similar low rates. A mutation in the large subunit of the replicative replication factor C complex (rfc1-1) increased the expansion rate for the (CGG)(25) repeat approximately 50-fold but had a much smaller effect on the expansion of the (CTG)(25) repeat. These data show dramatic sequence-specific expansion effects due to a mutation in the lagging strand DNA synthesis machinery. Together, the results of this study suggest that expansions are likely to result when the replication fork attempts to escape from the stall site.     

Isolation of CAG/CTG repeats from within the chromosome 2p21-p24 locus for autosomal dominant spastic paraplegia (SPG4) by YAC fragmentation

Pure autosomal dominant spastic paraplegia (SPG) is a genetically heterogeneous neurodegenerative disorder of the central nervous system clinically characterized by progressive spasticity mainly affecting the lower limbs. Three distinct loci have been mapped to chromosomes 14q (SPG3), 2p (SPG4) and 15q (SPG6). In particular, SPG4 families show striking intrafamilial variability suggestive of anticipation and evidence has been provided that CAG/CTG repeat expansions may be involved. To isolate CAG/CTG repeat containing sequences from within the SPG4 candidate region, a novel approach was developed. Fragmentation vectors were assembled allowing direct fragmentation of yeast artificial chromosomes (YACs) with a short (> or = 21 bp) CAG/CTG sequence as the target site for homologous recombination. We used the CAG/CTG YAC fragmentation vectors to isolate CAG/CTG containing sequences from four YACs spanning the SPG4 candidate region between D2S400 and D2S367. A total of four CAG/CTG containing sequences were isolated of which three were novel. However, none of the four CAG/CTG repeats showed expanded alleles in two Belgian SPG4 families. In addition, we showed that the CAG/CTG alleles detected by the repeat expansion detection (RED) method could be fully explained by two polymorphic nonpathogenic CAG/CTG repeats on chromosomes 17 and 18, respectively. Also, the RED expansions in six SPG families could not be explained by amplification of the CAG/CTG repeats at the SPG4 locus. Together, our data do not support the hypothesis of a CAG/CTG repeat expansion as the molecular mechanism underlying SPG4 pathology.     

Detection of triplet repeat expansion in the human genome by use of hybridization signal intensity

Triplet repeat disease is a group of hereditary neurodegenerative disorders caused by expansion of trinucleotide repeats such as CAG/CTG, CGG/CCG, and GAA/TTC. Direct detection of the expansion in the patient's genome shortcuts the tedious process needed for identification of disease genes by conventional approaches. Here we describe a method to detect triplet repeat expansion from the hybridization signal intensity. Using a digoxigenin-labeled (CTG)9 probe, the hybridization intensity and number of repeats showed a good linear correlation. The technique detected expansion in genomic DNA in all cases with moderate or large expansion. Even in the case of a small expansion, this method could detect the mutant fragment. The technique has advantages over related techniques because it is more sensitive and can be applied to cases where a small repeat expansion is involved.     

Identification of RTG2 as a modifier gene for CTG*CAG repeat instability in Saccharomyces cerevisiae

Trinucleotide repeats (TNRs) undergo frequent mutations in families affected by TNR diseases and in model organisms. Much of the instability is conferred in cis by the sequence and length of the triplet tract. Trans-acting factors also modulate TNR instability risk, on the basis of such evidence as parent-of-origin effects. To help identify trans-acting modifiers, a screen was performed to find yeast mutants with altered CTG.CAG repeat mutation frequencies. The RTG2 gene was identified as one such modifier. In rtg2 mutants, expansions of CTG.CAG repeats show a modest increase in rate, depending on the starting tract length. Surprisingly, contractions were suppressed in an rtg2 background. This creates a situation in a model system where expansions outnumber contractions, as in humans. The rtg2 phenotype was apparently specific for CTG.CAG repeat instability, since no changes in mutation rate were observed for dinucleotide repeats or at the CAN1 reporter gene. This feature sets rtg2 mutants apart from most other mutants that affect genetic stability both for TNRs and at other DNA sequences. It was also found that RTG2 acts independently of its normal partners RTG1 and RTG3, suggesting a novel function of RTG2 that helps modify CTG.CAG repeat mutation risk.     

Haploinsufficiency of yeast FEN1 causes instability of expanded CAG/CTG tracts in a length-dependent manner

Trinucleotide repeat diseases, such as Huntington's disease, are caused by the expansion of trinucleotide repeats above a threshold of about 35 repeats. Once expanded, the repeats are unstable and tend to expand further both in somatic cells and during transmission, resulting in a more severe disease phenotype. Flap endonuclease 1 (Fen1), has an endonuclease activity specific for 5' flap structures and is involved in Okazaki fragment processing and base excision repair. Fen1 also plays an important role in preventing instability of CAG/CTG trinucleotide repeat sequences, as the expansion frequency of CAG/CTG repeats is increased in FEN1 mutants in vitro and in yeast cells defective for the yeast homolog, RAD27. Here we have tested whether one copy of yeast FEN1 is enough to maintain CAG/CTG tract stability in diploid yeast cells. We found that CAG/CTG repeats are stable in RAD27 +/- cells if the tract is 70 repeats long and exhibit a slightly increased expansion frequency if the tract is 85 or 130 repeats long. However for CAG-155 tracts, the repeat expansion frequency in RAD27 +/- cells is significantly higher than in RAD27 +/+ cells. This data indicates that cells containing longer CAG/CTG repeats need more Fen1 protein to maintain tract stability and that maintenance of long CAG/CTG repeats is particularly sensitive to Fen1 levels. Our results may explain the relatively small effects seen in the Huntington's disease (HD) FEN1 +/- heterozygous mice and myotonic dystrophy type 1 (DM1) FEN1 +/- heterozygous mice, and suggest that inefficient flap processing by Fen1 could play a role in the continued expansions seen in humans with trinucleotide repeat expansion diseases.     

Replication inhibitors modulate instability of an expanded trinucleotide repeat at the myotonic dystrophy type 1 disease locus in human cells

Gene-specific CTG/CAG repeat expansion is associated with at least 14 human diseases, including myotonic dystrophy type 1 (DM1). Most of our understanding of trinucleotide instability is from nonhuman models, which have presented mixed results, supporting replication errors or processes independent of cell division as causes. Nevertheless, the mechanism occurring at the disease loci in patient cells is poorly understood. Using primary fibroblasts derived from a fetus with DM1, we have shown that spontaneous expansion of the diseased (CTG)(216) allele occurred in proliferating cells but not in quiescent cells. Expansions were "synchronous," with mutation frequencies approaching 100%. Furthermore, cells were treated with agents known to alter DNA synthesis but not to directly damage DNA. Inhibiting replication initiation with mimosine had no effect upon instability. Inhibiting both leading- and lagging-strand synthesis with aphidicolin or blocking only lagging strand synthesis with emetine significantly enhanced CTG expansions. It was striking that only the expanded DM1 allele was altered, leaving the normal allele, (CTG)(12), and other repeat loci unaffected. Standard and small-pool polymerase chain reaction revealed that inhibitors enhanced the magnitude of short expansions in most cells threefold, whereas 11%-25% of cells experienced gains of 122-170 repeats, to sizes of (CTG)(338)-(CTG)(386). Similar results were observed for an adult DM1 cell line. Our results support a role for the perturbation of replication fork dynamics in DM1 CTG expansions within patient fibroblasts. This is the first report that repeat-length alterations specific to a disease allele can be modulated by exogenously added compounds.     

Mutagenic stress modulates the dynamics of CTG repeat instability associated with myotonic dystrophy type 1

The molecular basis of the myotonic dystrophy type 1 is the expansion of a CTG repeat at the DMPK locus. The expanded disease-associated repeats are unstable in both somatic and germ lines, with a high tendency towards expansion. The rate of expansion is directly related to the size of the pathogenic allele, increasing the size heterogeneity with age. It has also been suggested that additional factors, including as yet unidentified environmental factors, might affect the instability of the expanded CTG repeats to account for the observed CTG size dynamics over time. To investigate the effect of environmental factors in the CTG repeat instability, three lymphoblastoid cell lines were established from two myotonic dystrophy patients and one healthy individual, and parallel cultures were concurrently expanded in the presence or absence of the mutagenic chemical mitomycin C for a total of 12 population doublings. The new alleles arising along the passages were analysed by radioactive small pool PCR and sequencing gels. An expansion bias of the stepwise mutation was observed in a (CTG)₁₂₄ allele of a cell line harbouring two modal alleles of 28 and 124 CTG repeats. Interestingly, this expansion bias was clearly enhanced in the presence of mitomycin C. The effect of mitomycin C was also evident in the normal size alleles in two cell lines with alleles of 13/13 and 12/69 repeats, where treated cultures showed new longer alleles. In conclusion, our results indicate that mitomycin C modulates the dynamics of myotonic dystrophy-associated CTG repeats in LBCLs, enhancing the expansion bias of long-pathogenic repeats and promoting the expansion of normal length repeats.     

Postreplication repair inhibits CAG.CTG repeat expansions in Saccharomyces cerevisiae

Trinucleotide repeats (TNRs) are unique DNA microsatellites that can expand to cause human disease. Recently, Srs2 was identified as a protein that inhibits TNR expansions in Saccharomyces cerevisiae. Here, we demonstrate that Srs2 inhibits CAG . CTG expansions in conjunction with the error-free branch of postreplication repair (PRR). Like srs2 mutants, expansions are elevated in rad18 and rad5 mutants, as well as the PRR-specific PCNA alleles pol30-K164R and pol30-K127/164R. Epistasis analysis indicates that Srs2 acts upstream of these PRR proteins. Also, like srs2 mutants, the pol30-K127/164R phenotype is specific for expansions, as this allele does not alter mutation rates at dinucleotide repeats, at nonrepeating sequences, or for CAG . CTG repeat contractions. Our results suggest that Srs2 action and PRR processing inhibit TNR expansions. We also investigated the relationship between PRR and Rad27 (Fen1), a well-established inhibitor of TNR expansions that acts at 5' flaps. Our results indicate that PRR protects against expansions arising from the 3' terminus, presumably replication slippage events. This work provides the first evidence that CAG . CTG expansions can occur by 3' slippage, and our results help define PRR as a key cellular mechanism that protects against expansions.     

SCA8 repeat expansion: large CTA/CTG repeat alleles are more common in ataxic patients,including those with SCA6

We analyzed the SCA8 CTA/CTG repeat in a large group of Japanese subjects. The frequency of large alleles (85-399 CTA/CTG repeats) was 1.9% in spinocerebellar ataxia (SCA), 0.4% in Parkinson disease, 0.3% in Alzheimer disease, and 0% in a healthy control group; the frequency was significantly higher in the group with SCA than in the control group. Homozygotes for large alleles were observed only in the group with SCA. In five patients with SCA from two families, a large SCA8 CTA/CTG repeat and a large SCA6 CAG repeat coexisted. Age at onset was correlated with SCA8 repeats rather than SCA6 repeats in these five patients. In one of these families, at least one patient showed only a large SCA8 CTA/CTG repeat allele, with no large SCA6 CAG repeat allele. We speculate that the presence of a large SCA8 CTA/CTG repeat allele influences the function of channels such as alpha(1A)-voltage-dependent calcium channel through changing or aberrant splicing, resulting in the development of cerebellar ataxia, especially in homozygous patients.     

A novel selectable system for detecting expansion of CAG.CTG repeats in mammalian cells

CAG.CTG repeat expansions cause more than a dozen neurodegenerative diseases in humans. To define the mechanism of repeat instability in mammalian cells we developed a selectable assay to detect expansions of CAG.CTG triplet repeats in Chinese hamster ovary (CHO) cells. We showed previously that long tracts of CAG.CTG repeats, embedded in an intron of the APRT gene, kill expression of the gene, rendering the cells APRT-. By contrast, tracts with fewer than 34 repeats allow sufficient expression to give APRT+ cells. Although it should be possible to use APRT+ cells with short repeats to assay for expansion events by selecting for APRT- cells, we find that APRT+ cells with 31 repeats are not killed by the standard APRT- selection protocol, most likely because they produce too little Aprt to incorporate sufficient 8-azaadenine into their adenine pool. To overcome this problem, we devised a new selection, which increases the proportion of the adenine pool contributed by the salvage pathway by partially inhibiting the de novo pathway. We show that APRT- CHO cells with 61 or 95 CAG.CTG repeats survive this selection, whereas cells with 31 repeats die. Using this selection system, we can select for expansion to as few as 39 repeats. Thus, this assay can monitor expansions across the critical boundary from the longest lengths of normal alleles to the shortest lengths of disease alleles.     

Disease-associated repeat instability and mismatch repair

Expanded tandem repeat sequences in DNA are associated with at least 40 human genetic neurological, neurodegenerative, and neuromuscular diseases. Repeat expansion can occur during parent-to-offspring transmission, and arise at variable rates in specific tissues throughout the life of an affected individual. Since the ongoing somatic repeat expansions can affect disease age-of-onset, severity, and progression, targeting somatic expansion holds potential as a therapeutic target. Thus, understanding the factors that regulate this mutation is crucial. DNA repair, in particular mismatch repair (MMR), is the major driving force of disease-associated repeat expansions. In contrast to its anti-mutagenic roles, mammalian MMR curiously drives the expansion mutations of disease-associated (CAG)·(CTG) repeats. Recent advances have broadened our knowledge of both the MMR proteins involved in disease repeat expansions, including: MSH2, MSH3, MSH6, MLH1, PMS2, and MLH3, as well as the types of repeats affected by MMR, now including: (CAG)·(CTG), (CGG)·(CCG), and (GAA)·(TTC) repeats. Mutagenic slipped-DNA structures have been detected in patient tissues, and the size of the slip-out and their junction conformation can determine the involvement of MMR. Furthermore, the formation of other unusual DNA and R-loop structures is proposed to play a key role in MMR-mediated instability. A complex correlation is emerging between tissues showing varying amounts of repeat instability and MMR expression levels. Notably, naturally occurring polymorphic variants of DNA repair genes can have dramatic effects upon the levels of repeat instability, which may explain the variation in disease age-of-onset, progression and severity. An increasing grasp of these factors holds prognostic and therapeutic potential.