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[Medical pathology due to trinucleotide repeats].

Trinucleotide repeat expansion is responsible for ten human diseases described so far. Four types of repeats are involved in these expansions, with type, number and position in the gene varying from one disease to another. In some fragile sites, the trinucleotide repeat is found to be enlarged to 200 or more. Smaller expansions have been found within coding regions of some genes that are associated with neurodegenerative diseases, such as Huntington's disease. The continuous expansion of the trinucleotide repeats in subsequent generations explains the genetic anticipation, peculiar to these disorders. Recently, it was shown that two expanded minisatellite sequences are also involved in both progressive myoclonus epilepsy type 1 and distamycin A-sensitive fragile site, FRA16B. This form of peculiar heredity is very important because of its relationship with some of the common human degenerative diseases.

Chromosome Aberrations↗

PubMed1999

Double-strand break repair can lead to high frequencies of deletions within short CAG/CTG trinucleotide repeats.

Trinucleotide repeats undergo contractions and expansions in humans, leading in some cases to fatal neurological disorders. The mechanism responsible for these large size variations is unknown, but replication-slippage events are often suggested as a possible source of instability. We constructed a genetic screen that allowed us to detect spontaneous expansions/contractions of a short trinucleotide repeat in yeast. We show that deletion of RAD27, a gene involved in the processing of Okazaki fragments, increases the frequency of contractions tenfold. Repair of a chromosomal double-strand break (DSB) using a trinucleotide repeat-containing template induces rearrangements of the repeat with a frequency 60 times higher than the natural rate of instability of the same repeat. Our data suggest that both gene conversion and single-strand annealing are major sources of trinucleotide repeat rearrangements.

Base Sequence↗

PubMed2004

Saccharomyces cerevisiae Srs2 DNA helicase selectively blocks expansions of trinucleotide repeats.

Trinucleotide repeats (TNRs) undergo frequent mutations in families afflicted with certain neurodegenerative disorders and in model organisms. TNR instability is modulated both by the repeat tract itself and by cellular proteins. Here we identified the Saccharomyces cerevisiae DNA helicase Srs2 as a potent and selective inhibitor of expansions. srs2 mutants had up to 40-fold increased expansion rates of CTG, CAG, and CGG repeats. The expansion phenotype was specific, as mutation rates at dinucleotide repeats, at unique sequences, or for TNR contractions in srs2 mutants were not altered. Srs2 is known to suppress inappropriate genetic recombination; however, the TNR expansion phenotype of srs2 mutants was largely independent of RAD51 and RAD52. Instead, Srs2 mainly functioned with DNA polymerase delta to block expansions. The helicase activity of Srs2 was important, because a point mutant lacking ATPase function was defective in blocking expansions. Purified Srs2 was substantially better than bacterial UvrD helicase at in vitro unwinding of a DNA substrate that mimicked a TNR hairpin. Disruption of the related helicase gene SGS1 did not lead to excess expansions, nor did wild-type SGS1 suppress the expansion phenotype of an srs2 strain. We conclude that Srs2 selectively blocks triplet repeat expansions through its helicase activity and primarily in conjunction with polymerase delta.

DNA Helicases↗

PubMed2005

Length-dependent energetics of (CTG)n and (CAG)n trinucleotide repeats.

Trinucleotide repeats are involved in a number of debilitating diseases such as myotonic dystrophy. Twelve to seventy-five base-long (CTG)n oligodeoxynucleotides were analysed using a combination of biophysical [UV-absorbance, circular dichroism and differential scanning calorimetry (DSC)] and biochemical methods (non-denaturing gel electrophoresis and enzymatic footprinting). All oligomers formed stable intramolecular structures under near physiological conditions with a melting temperature that was only weakly dependent on oligomer length. Thermodynamic analysis of the denaturation process by UV-melting and calorimetric experiments revealed an unprecedented length-dependent discrepancy between the enthalpy values deduced from model-dependent (UV-melting) and model-independent (calorimetry) experiments. Evidence for non-zero molar heat capacity changes was also derived from the analysis of the Arrhenius plots and DSC profiles. Such behaviour is analysed in the framework of an intramolecular 'branched-hairpin' model, in which long CTG oligomers do not fold into a simple long hairpin-stem intramolecular structure, but allow the formation of several independent folding units of unequal stability. We demonstrate that, for sequences ranging from 12 to 25 CTG repeats, an intramolecular structure with two loops is formed which we will call 'bis-hairpin'. Similar results were also found for CAG oligomers, suggesting that this observation may be extended to various trinucleotide repeats-containing sequences.

Base Sequence↗

PubMed2006

Length and pH-dependent energetics of (CCG)n and (CGG)n trinucleotide repeats.

Trinucleotide repeats are involved in a number of debilitating diseases such as fragile-X syndrome and myotonic dystrophy. Eighteen to 75 base-long (CCG)(n) and (CGG)(n) oligodeoxynucleotides were analysed using a combination of biophysical (UV-absorbance, differential scanning calorimetry) and biochemical methods (non-denaturing gel electrophoresis, enzymatic footprinting). All oligomers formed stable intramolecular structures under near physiological conditions with a melting temperature which was only weakly dependent on oligomer length. Thermodynamic analysis of the denaturation process by UV-melting and calorimetric experiments revealed a length-dependent discrepancy between the enthalpy values deduced from model-dependent (UV-melting) and model-independent experiments (calorimetry), as recently shown for CTG and CAG trinucleotides (Nucleic Acids Res. 33 (2005) 4065). Evidence for non-zero molar heat capacity changes was also derived from the analysis of the Arrhenius plots. Such behaviour is analysed in the framework of an intramolecular "branched" or "broken" hairpin model, in which long oligomers do not fold into a simple long hairpin-stem intramolecular structure, but allow the formation of several independent folding units of unequal stability. These results suggest that this observation may be extended to various trinucleotide repeats-containing sequences.

Base Sequence↗

PubMed1998

Ribozyme-mediated trans-splicing of a trinucleotide repeat.

Trinucleotide repeat expansions (TREs) are a recently described class of mutations characterized by a change in the size of the genomic fragment due to amplification of the repeated unit. A number of diseases have been attributed to TRE, including Huntington disease and myotonic dystrophy (DM), but attempts at genetic therapy have yet to prove successful. A potential therapeutic approach would be to repair the expanded repeat using the trans-splicing ability of group I intron ribozymes. We have used DM as a model to test this hypothesis. A group I intron ribozyme (DMPK-RZ1) was designed to modify the TRE at the 3' end of the human myotonic dystrophy protein kinase (DMPK) transcripts. DMPK-RZ1 was shown to ligate a small DMPK mRNA fragment, contained within the ribozyme, to a simple DMPK-target RNA in vitro. It also modified a larger target transcript, leading to replacement of twelve repeats with five repeats, both in vitro and in mammalian cells. Finally, this ribozyme successfully replaced the 3' end of endogenous DMPK mRNA in fibroblasts with a different 3' region. Ribozyme-mediated RNA repair may thus form a novel therapeutic strategy for diseases associated with repeat expansions.

Cell Line↗

PubMed2003

[Methods of determination of the number of CTG/CAG repeats in trinucleotide repeats in the human genome].

BACKGROUND: Human genome dynamic mutations are a new class of gene mutations represented by an unstable number of trinucleotide repeats and causing severe human hereditary neuromuscular and neurodegenerative diseases. The identification of pathological expanded alleles on the molecular level is important for clinical diagnostics. METHODS AND RESULTS: For the molecular diagnostics of expanded tandem repeat trinucleotide sequences we have introduced a fast and efficient TP-PCR fluorescent method according to Warner et al. (1996). We have modified this TP-PCR method for a rapid detection of expanded CTG alleles of the DMPK gene (myotonic dystrophy, MD) into a two-level protocol; first, the heterozygote sample DNAs were selected using P1/P2 primers flanking repeat tracts and, second, the TP-PCR protocol used was focused above all on the identification of a pathological allele. A fluorescent-labelled specific primer in TP-PCR was used for the exact determination of the number of CAG repeats of the gene IT-15 (Huntington's disease--HD) in the diagnostically important region of the grey zone (35 to 39 CAG). The reproducibility of the PCR results was demonstrated on control DNA samples with the known genotype and, in the case of MD, also by Southern blot analysis. We have especially shown the possibility of a cheaper PCR-P1/P2 and TP-PCR protocol, which can be used, with silver staining of separated PCR products on polyacrylamide gels. CONCLUSIONS: Our experience with introducing the above-mentioned PCR methods into laboratory practice clearly documents the possibilities of their general applicability in the molecular diagnostics of hereditary diseases characterised by instability of the trinucleotide repeat tracts.

Humans↗

PubMed1995

Population genetics of trinucleotide repeat polymorphisms.

Trinucleotide repeats at five disease loci (DM, DRPLA, HD, SBMA and SCA1) were surveyed in phenotypically normal individuals from three continental populations. This is the first analysis to examine the population dynamics of these five disease-related trinucleotide repeats in the same individuals from worldwide populations. Roughly half of all alleles observed at each locus are shared between all continental groups. For three loci, disease prevalence in each population corresponds with the number of alleles in the upper tail of the allele-size distribution. The allele-size distributions of African, Asian and Caucasian groups show a high degree of variation, and gene diversity estimates for trinucleotide repeat loci exceed estimates derived from dinucleotide or tetranucleotide repeats. Analyses that compared infinite alleles and stepwise mutation models suggest that normal variation at trinucleotide loci is not generated by stepwise mutation alone. Trees constructed for subpopulations using trinucleotide repeat loci show accurate continental clustering. Interpopulation genetic distance estimates show remarkable similarity to distance estimates produced from tetranucleotide repeats or nuclear restriction site polymorphisms. This finding is especially noteworthy in light of the fact that trinucleotide repeat polymorphisms at these loci can cause disease, while restriction site and tetranucleotide polymorphisms appear to be selectively neutral. In contrast, genetic distance estimates from trinucleotide loci are poorly correlated with genetic distance estimates from mitochondrial sequence data.

Alleles↗

PubMed2006

Tandemly repeated trinucleotides - comparative analysis.

Characteristics of 64 possible tandem trinucleotide repeats (TSSR) from Homo sapiens (hs), Mus musculus (mm) and Rattus norvegicus (rn) genomes are presented. Comparative analysis of TSSR frequency depending on their repetitiveness and similarity of the TSSR length distributions is shown. Comparative analysis of TSSR sequence motifs and association between type of motif and its length (n) using rho-coefficient method (quantitatively measuring the association between variables in contingency tables) is presented. These analyses were carried out in the context of neurodegenerative diseases based on trinucleotide tandems. The length of these tandems and their relation to other TSSR is estimated. It was found that the higher repetitiveness (n) the lower frequency of trinucleotides tandems. Differences between genomes under consideration, especially in longer than n=9 TSSR were discussed. A significantly higher frequency off A- and T-rich tandems is observed in the human genome (as well as in human mRNA). This observation also applies to mm and rn, although lower abundant in proportion to human genomes was found. The origin of elongation (or shortening) of TSSR seems to be neither frequency nor length dependent. The results of TSSR analysis presented in this work suggest that neurodegenerative disease-related microsatellites do not differ versus the other except the lower frequency versus the other TSSR. CAG occurs with relatively high frequency in human mRNA, although there are other TSSR with higher frequency that do not cause comparable disease disorders. It suggests that the mechanism of TSSR instability is not the only origin of neurodegenerative diseases.

Animals↗

PubMed2005

Small-molecule ligand induces nucleotide flipping in (CAG)n trinucleotide repeats.

DNA trinucleotide repeats, particularly CXG, are common within the human genome. However, expansion of trinucleotide repeats is associated with a number of disorders, including Huntington disease, spinobulbar muscular atrophy and spinocerebellar ataxia. In these cases, the repeat length is known to correlate with decreased age of onset and disease severity. Repeat expansion of (CAG)n, (CTG)n and (CGG)n trinucleotides may be related to the increased stability of alternative DNA hairpin structures consisting of CXG-CXG triads with X-X mismatches. Small-molecule ligands that selectively bound to CAG repeats could provide an important probe for determining repeat length and an important tool for investigating the in vivo repeat extension mechanism. Here we report that napthyridine-azaquinolone (NA, 1) is a ligand for CAG repeats and can be used as a diagnostic tool for determining repeat length. We show by NMR spectroscopy that binding of NA to CAG repeats induces the extrusion of a cytidine nucleotide from the DNA helix.

DNA↗

PubMed2002

Transcription and nuclear transport of CAG/CTG trinucleotide repeats in yeast.

Trinucleotide repeats are involved in several neurological disorders in humans. DNA sequences containing CAG/CTG repeats are prone to slippage during replication and double-strand break repair. The effects of trinucleotide repeats on transcription and on nuclear export were analyzed in vivo in yeast. Transcription of a CAG/CTG trinucleotide repeat in the 3'-untranslated region of a URA3 reporter gene leads to transcription of messenger RNAs several kilobases longer than the expected size. These long mRNAs form more readily when CAG rather than CTG repeats are transcribed. CAG- or CUG-containing transcripts show a non-homogeneous cellular localization. We propose that long mRNAs result from transcription slippage, and discuss the possible implications for human diseases.

3' Untranslated Regions↗

PubMed2004

The effect of pre-mutation of X chromosome CGG trinucleotide repeats on brain anatomy.

Expanded trinucleotide repeats are associated with several neuropsychiatric disorders, including fragile X syndrome (FraX) which is the most common inherited form of mental retardation. It is currently thought that FraX results from having >200 CGG trinucleotide repeats, with consequent methylation of the fragile X mental retardation gene (FMR1) and loss of FMR1 protein (FMRP). Pre-mutation carriers of FraX (with 55-200 CGG trinucleotide repeats) were originally considered unaffected, although recent studies challenge this view. However, there are few studies on the effect of pre-mutation trinucleotide repeat expansion on the male human brain using quantitative MRI. Also the results of prior investigations may be confounded because people were selected on the basis of clinical and neurological features, and not genetic phenotype. We compared the brain anatomy of 20 adult male pre-mutation members of known FraX families with 20 healthy male controls. The two groups did not differ significantly in age, intelligence quotient (IQ) or handedness. We also investigated whether any observed effects were associated with: (i) ageing; (ii) expansion of pre-mutation CGG trinucleotide repeats; (iii) reduction in the percentage of lymphocytes staining with anti-FMRP antibodies [%FMRP(+) lymphocytes]; and (iv) elevation of FMR1 mRNA levels. Male pre-mutation carriers of FraX, compared with matched controls, had significantly less voxel density in several brain regions, including the cerebellum, amygdalo-hippocampal complex and thalamus. Within pre-mutation carriers of FraX, ageing, increases in the number of CGG trinucleotide repeats and decreases in %FMRP(+) lymphocytes were associated with decreasing voxel density of regions previously identified as decreased relative to controls. Regional grey and white matter density is significantly affected in male pre-mutation carriers of FraX recruited on the basis of genetic, not clinical, phenotype. The association of voxel density reduction and ageing is consistent with observations of a subgroup of older pre-mutation males who present with cognitive decline. Moreover, our findings suggest, for the first time, an association between voxel density reduction and genetic variation in FraX.

Adolescent↗

PubMed1998

Rapid cloning of expanded trinucleotide repeat sequences from genomic DNA.

Trinucleotide repeat expansions have been shown to cause a number of neurodegenerative diseases. A hallmark of most of these diseases is the presence of anticipation, a decrease in the age at onset in consecutive generations due to the tendency of the unstable trinucleotide repeat to lengthen when passed from one generation to the next. The involvement of trinucleotide repeat expansions in a number of other diseases--including familial spastic paraplegia, schizophrenia, bipolar affective disorder and spinocerebellar ataxia type 7 (SCA7; ref. 10)--is suggested both by the presence of anticipation and by repeat expansion detection (RED) analysis of genomic DNA samples. The involvement of trinucleotide expansions in these diseases, however, can be conclusively confirmed only by the isolation of the expansions present in these populations and detailed analysis to assess each expansion as a possible pathogenic mutation. We describe a novel procedure for quick isolation of expanded trinucleotide repeats and the corresponding flanking nucleotide sequence directly from small amounts of genomic DNA by a process of Repeat Analysis, Pooled Isolation and Detection of individual clones containing expanded trinucleotide repeats (RAPID cloning). We have used this technique to clone the pathogenic SCA7 CAG expansion from an archived DNA sample of an individual affected with ataxia and retinal degeneration.

Ataxia↗

PubMed1997

Trinucleotide repeats in the human genome: size distributions for all possible triplets and detection of expanded disease alleles in a group of Huntington disease individuals by the repeat expansion detection method.

Using a modified Repeat Expansion Detection (RED) assay, that was optimized for individual oligonucleotides, unrelated individuals were systematically screened for maximal repeat sizes of each of the ten possible trinucleotide repeats. Cloned trinucleotide repeats were generated and used as standards for the detectability of single copy trinucleotide repeat fragments. When the size distributions of trinucleotide repeats were compared to previously reported data, significant differences were found for the CTT repeat, which corresponds to the expanded GAA repeat in Friedreich ataxia, as well as for ATT, CCT and GTT repeats. Since 30-35% of normal individuals have CTG/CAG trinucleotide repeat sizes of 180 bp or more, we investigated the question whether small-scale CTG/CAG repeat expansions are detectable on a population basis by using the RED technique. We blindly screened 20 HD probands with CAG expansions of the HD gene, ranging in size between 120 and 174 bp, and found that a shift to larger CAG size ranges is clearly detectable when comparing the distribution of maximal repeat sizes in the disease group to a control group. Our study, therefore, demonstrates that the application of the RED assay to a population of probands and a population of controls allows the detection of small-scale CTG/CAG repeat expansions in the size range of the expanded HD gene and present in a single allele. We also provide standards and control data for the detection of other trinucleotide repeat expansions.

Adult↗

PubMed1997

The complex pathology of trinucleotide repeats.

The expansion of trinucleotide repeat sequences has now been shown to be the underlying cause of at least ten human disorders. Unifying features among these diseases include the unstable behavior of the triplet repeat during germline transmission when the length of the repeat exceeds a critical value. However, the trinucleotide repeat disorders can be divided into two distinct groups. Type I disorders involve the expansion of CAG repeats, which encode an expanded polyglutamine, inserted into the open-reading frame of a gene that is usually quite broadly expressed. Recently, mouse models for type I disorders have been developed and the basis of pathology is under study, both in these models and through biochemical and cell biological approaches. The type II disorders involve repeat expansions in noncoding regions of genes. The mechanisms by which these repeat expansions lead to pathology may be quite diverse.

Animals↗

PubMed1997

Ocular changes in patients with spinocerebellar degeneration and repeated trinucleotide expansion of spinocerebellar ataxia type 1 gene.

OBJECTIVE: To examine ocular changes in patients with spinocerebellar degeneration who have repeated trinucleotide expansion in the spinocerebellar ataxia type 1 (SCA1) gene. DESIGN: Ophthalmic findings in 6 patients from 3 families whose DNA analysis revealed that they had an expanded allele of the trinucleotide repeated in the SCA1 gene were compared with those of normal control subjects and other healthy family members. The DNA was extracted from peripheral blood lymphocytes of the neurodegenerative family and normal control subjects. SETTING: University medical center. RESULTS: Visual acuity gradually decreased in successive follow-up visits. Color vision and visual fields were gradually affected. Electroretinograms showed mild attenuation of oscillatory potentials. Corneal endothelial cell density was severely decreased from 600 to 1300 cells/mm2. These findings were not observed in the normal control subjects, other healthy family members, or other patients with spinocerebellar degeneration who had repeated trinucleotide expansion of other genes. CONCLUSION: To the best of our knowledge, this is the first report describing the association between ocular changes in patients with spinocerebellar degeneration and gene mutation. These ocular changes were considered specific to patients who had the expanded allele of the repeated trinucleotide in the SCA1 gene.

Adult↗

PubMed1997

Trinucleotide repeat instability: genetic features and molecular mechanisms.

Trinucleotide repeat expansions are an important cause of inherited neurodegenerative disease. The expanded repeats are unstable, changing in size when transmitted from parents to offspring (intergenerational instability, "meiotic instability") and often showing size variation within the tissues of an affected individual (somatic mosaicism, "mitotic instability"). Repeat instability is a clinically important phenomenon, as increasing repeat lengths correlate with an earlier age of onset and a more severe disease phenotype. The tendency of expanded trinucleotide repeats to increase in length during their transmission from parent to offspring in these diseases provides a molecular explanation for anticipation (increasing disease severity in successive affected generations). In this review, I explore the genetic and molecular basis of trinucleotide repeat instability. Studies of patients and families with trinucleotide repeat disorders have revealed a number of factors that determine the rate and magnitude of trinucleotide repeat change. Analysis of trinucleotide repeat instability in bacteria, yeast, and mice has yielded additional insights. Despite these advances, the pathways and mechanisms underlying trinucleotide repeat instability in humans remain largely unknown. There are many reasons to suspect that this uniquely human phenomenon will significantly impact upon our understanding of development, differentiation and neurobiology.

Animals↗

PubMed1996

Trinucleotide repeats in neurogenetic disorders.

Trinucleotide repeat expansion is increasingly recognized as a cause of neurogenetic diseases. To date, seven diseases have been identified as expanded repeat disorders: the fragile X syndrome of mental retardation both FRAXA and FRAXE loci), myotonic dystrophy, X-linked spinal and bulbar muscular atrophy, Huntington's disease, spinocerebellar ataxia type I, dentatorubral-pallidoluysian atrophy, and Machado-Joseph disease. All are neurologic disorders, affecting one or more regions of the neuraxis. Moreover, five of the seven (the last five above) are progressive neurodegenerative disorders whose strikingly similar mutations suggest a common mechanism of neuronal degeneration. In this article we discuss specific characteristics of each trinucleotide repeat disease, review their shared clinical and genetic features, and address possible molecular mechanisms underlying the neuropathology in each disease. Particular attention is paid to the neurodegenerative diseases, all of which are caused by CAG repeats encoding polyglutamine tracts in the disease gene protein.

Central Nervous System Diseases↗