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Results for “DNA Repeat Expansion”

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At least 163 records · Page 9Linked to original sources
PubMed2004

Modified single-stranded oligonucleotides inhibit aggregate formation and toxicity induced by expanded polyglutamine.

Huntington's disease (HD) is caused by an increase in the length of the poly(Q) tract in the huntingtin (Htt) protein, which changes its solubility and induces aggregation. Aggregation occurs in two general phases, nucleation and elongation, and agents designed to block either phase are being considered as potential therapeutics. We demonstrate that inclusion formation can be retarded by introducing modified, single-stranded oligonucleotides into a model neuronal cell line. This cell-based assay is used in conjunction with a standardized biochemical assay to identify molecules that can disrupt the process of aggregate formation. Active oligonucleotides include a 6-mer containing a single phosphorothioate linkage on each terminus, a 53-mer and a 9-mer containing three phosphorothioate linkages at each end, and a 25-mer consisting of all modified RNA residues. The disruption process directed by the active oligonucleotides appears to be independent of sequence specificity and complementarity. In contrast, the activity is more dependent on the type of chemical modifications contained within the oligonucleotide. Some oligonucleotides that demonstrated inhibition activity were also found to extend the life span of PC12 cells after the toxic Htt aggregation process was induced. Our data provide the first evidence that short synthetic oligonucleotides inhibit a fundamental pathological pathway of HD and may provide the basis for a novel therapeutic approach.

Animals↗
PubMed2004

Recovery from polyglutamine-induced neurodegeneration in conditional SCA1 transgenic mice.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant, polyglutamine-induced neurodegenerative disorder that results in loss of motor coordination caused primarily by a disruption of cerebellar Purkinje cell function. In this study, we developed a conditional SCA1 mouse model to examine whether stopping expression of mutant ataxin-1 alters the disease phenotype. After cessation of SCA1[82Q] transgene expression, mutant ataxin-1, including that in nuclear inclusions, was cleared rapidly from Purkinje cells. At an early stage of disease, Purkinje cell pathology and motor dysfunction were completely reversible. After halting SCA1 expression at later stages of disease, only a partial recovery was seen. Interestingly, restoration of the ability to perform a complex motor task, the accelerating Rotarod, correlated with localization of mGluR1alpha to the Purkinje cell-parallel fiber synapse. These results show that the progression of SCA1 pathogenesis is dependent on the continuous expression of mutant ataxin-1. Of note, even at a late stage of disease, Purkinje cells retain at least some ability to repair the damage caused by mutant ataxin-1.

Animals↗
PubMed2003

B chromosomes of the Podisma sapporensis Shir. (Orthoptera, Acrididae) analysed by chromosome microdissection and FISH.

The analysis of the distribution of repetitive DNA of the B chromosomes of Podisma sapporensis in the A and B chromosomes of the natural populations and in A chromosomes of three other species of the Podismini grasshoppers were made. DNA-libraries of the B chromosome and the euchromatic segment of the A chromosome of P. sapporensis were generated by meiotic chromosome microdissection followed by degenerated oligonucleotide primed polymerase chain reaction (DOP-PCR). Paints based on these DNA-libraries were used for FISH analysis to detect localization of homologous sequences in A and B chromosomes of P. sapporensis from different natural populations. On the basis of the FISH analysis the authors suggest that evolution of the B chromosomes in Podisma sapporensis was associated mainly with the insertions of "alien DNA sequences" into ancestral A chromosome and their further amplification. The number of initial sites of amplifications differed in the different Bs, the distance between these sites also varying. Karyotype evolution in P. sapporensis was associated partly with the insertion of "alien DNA sequences" into pericentromeric chromosomal regions. Insertion into the small short arms of the acrocentric chromosomes followed, with the DNA amplification leading to the formation of the additional C-heterochromatic arms or euchromatic-like regions of different size.

Animals↗
PubMed2002

Conformational energetics of stable and metastable states formed by DNA triplet repeat oligonucleotides: implications for triplet expansion diseases.

We have embedded the hexameric triplet repeats (CAG)(6) and (CTG)(6) between two (GC)(3) domains to produce two 30-mer hairpins with the sequences d[(GC)(3)(CAG)(6)(GC)(3)] and d[(GC)(3)(CTG)(6)(GC)(3)]. This construct reduces the conformational space available to these repetitive DNA sequences. We find that the (CAG)(6) and (CTG)(6) repeats form stable, ordered, single-stranded structures. These structures are stabilized at 62 degrees C by an average enthalpy per base of 1.38 kcal.mol(-1) for the CAG triplet and 2.87 kcal.mol(-1) for the CTG triplet, while being entropically destabilized by 3.50 cal.K(-1).mol(-1) for the CAG triplet and 7.6 cal.K(-1).mol(-1) for the CTG triplet. Remarkably, these values correspond, respectively, to 1/3 (for CAG) and 2/3 (for CTG) of the enthalpy and entropy per base values associated with Watson-Crick base pairs. We show that the presence of the loop structure kinetically inhibits duplex formation from the two complementary 30-mer hairpins, even though the duplex is the thermodynamically more stable state. Duplex formation, however, does occur at elevated temperatures. We propose that this thermally induced formation of a more stable duplex results from thermal disruption of the single-stranded order, thereby allowing the complementary domains to associate (perhaps via "kissing hairpins"). Our melting profiles show that, once duplex formation has occurred, the hairpin intermediate state cannot be reformed, consistent with our interpretation of kinetically trapped hairpin structures. The duplex formed by the two complementary oligonucleotides does not have any unusual optical or thermodynamic properties. By contrast, the very stable structures formed by the individual single-stranded triplet repeat sequences are thermally and thermodynamically unusual. We discuss this stable, triplet repeat, single-stranded structure and its interconversion with duplex in terms of triplet expansion diseases.

Base Sequence↗
PubMed1992

DNA structure, mutations, and human genetic disease.

The etiology of fragile X syndrome, myotonic dystrophy and Kennedy's disease has been attributed to the massive expansion of triplet repeat DNA sequences. This review details the relationships between the structural diversity of DNA, its secondary structure or DNA-directed mutagenesis, and the expansion of triplet repeats.

Base Sequence↗
PubMed2005

Interactions among DNA ligase I, the flap endonuclease and proliferating cell nuclear antigen in the expansion and contraction of CAG repeat tracts in yeast.

Among replication mutations that destabilize CAG repeat tracts, mutations of RAD27, encoding the flap endonuclease, and CDC9, encoding DNA ligase I, increase the incidence of repeat tract expansions to the greatest extent. Both enzymes bind to proliferating cell nuclear antigen (PCNA). To understand whether weakening their interactions leads to CAG repeat tract expansions, we have employed alleles named rad27-p and cdc9-p that have orthologous alterations in their respective PCNA interaction peptide (PIP) box. Also, we employed the PCNA allele pol30-90, which has changes within its hydrophobic pocket that interact with the PIP box. All three alleles destabilize a long CAG repeat tract and yield more tract contractions than expansions. Combining rad27-p with cdc9-p increases the expansion frequency above the sum of the numbers recorded in the individual mutants. A similar additive increase in tract expansions occurs in the rad27-p pol30-90 double mutant but not in the cdc9-p pol30-90 double mutant. The frequency of contractions rises in all three double mutants to nearly the same extent. These results suggest that PCNA mediates the entry of the flap endonuclease and DNA ligase I into the process of Okazaki fragment joining, and this ordered entry is necessary to prevent CAG repeat tract expansions.

Alleles↗
PubMed1999

Transcription factor UAF, expansion and contraction of ribosomal DNA (rDNA) repeats, and RNA polymerase switch in transcription of yeast rDNA.

Strains of the yeast Saccharomyces cerevisiae defective in transcription factor UAF give rise to variants able to grow by transcribing endogenous ribosomal DNA (rDNA) by RNA polymerase II (Pol II). We have demonstrated that the switch to growth using the Pol II system consists of two steps: a mutational alteration in UAF and an expansion of chromosomal rDNA repeats. The first step, a single mutation in UAF, is sufficient to allow Pol II transcription of rDNA. In contrast to UAF mutations, mutations in Pol I or other Pol I transcription factors can not independently lead to Pol II transcription of rDNA, suggesting a specific role of UAF in preventing polymerase switch. The second step, expansion of chromosomal rDNA repeats to levels severalfold higher than the wild type, is required for efficient cell growth. Mutations in genes that affect recombination within the rDNA repeats, fob1 and sir2, decrease and increase, respectively, the frequency of switching to growth using Pol II, indicating that increased rDNA copy number is a cause rather than a consequence of the switch. Finally, we show that the switch to the Pol II system is accompanied by a striking alteration in the localization and morphology of the nucleolus. The altered state that uses Pol II for rDNA transcription is semistable and heritable through mitosis and meiosis. We discuss the significance of these observations in relation to the plasticity of rDNA tandem repeats and nucleolar structures as well as evolution of the Pol I machinery.

Adaptation, Physiological↗
PubMed1994

Preferential nucleosome assembly at DNA triplet repeats from the myotonic dystrophy gene.

The expansion of CTG repeats in DNA occurs in or near genes involved in several human diseases, including myotonic dystrophy and Huntington's disease. Nucleosomes, the basic structural element of chromosomes, consist of 146 base pairs of DNA coiled about an octamer of histone proteins and mediate general transcriptional repression. Electron microscopy was used to examine in vitro the nucleosome assembly of DNA containing repeating CTG triplets. The efficiency of nucleosome formation increased with expanded triplet blocks, suggesting that such blocks may repress transcription through the creation of stable nucleosomes.

DNA↗
PubMed2004

Length-dependent structure formation in Friedreich ataxia (GAA)n*(TTC)n repeats at neutral pH.

More than 15 human genetic diseases have been associated with the expansion of trinucleotide DNA repeats, which may involve the formation of non-duplex DNA structures. The slipped-strand nucleation of duplex DNA within GC-rich trinucleotide repeats may result in the changes of repeat length; however, such a mechanism seems less likely for the AT-rich (GAA)n*(TTC)n repeats. Using two-dimensional agarose gels, chemical probing and atomic force microscopy, we characterized the formation of non-B-DNA structures in the Friedreich ataxia-associated (GAA)n*(TTC)n repeats from the FRDA gene that were cloned with flanking genomic sequences into plasmids. For the normal genomic repeat length (n = 9) our data are consistent with the formation of a very stable protonated intramolecular triplex (H-DNA). Its stability at pH 7.4 is likely due to the high proportion of the T.A.T triads which form within the repeats as well as in the immediately adjacent AT-rich sequences with a homopurine. homopyrimidine bias. At the long normal repeat length (n = 23), a family of H-DNAs of slightly different sizes has been detected. At the premutation repeat length (n = 42) and higher negative supercoiling, the formation of a single H-DNA structure becomes less favorable and the data are consistent with the formation of a bi-triplex structure.

AT Rich Sequence↗
PubMed2000

[Satellite DNA and disease--possible mechanisms. Trinucleotide repeats].

The new data on the mechanisms underlying trinucleotide repeat expansion are reviewed with special reference to the role of chromatin structure, DNA replication, methylation, and amplification in repeat expansion during ontogeny. A hypothesis is advanced as to the crucial role of processes that occur at the preimplantation developmental stage, such as sister chromatid exchanges, single-strand DNA breaks, and demethylation. The molecular and cellular events responsible for association between trinucleotide expansion and various diseases are discussed.

DNA, Satellite↗
PubMed2003

Huntingtin contains a highly conserved nuclear export signal.

Huntington's disease (HD), is a genetic neurodegenerative disease characterized by a DNA CAG triplet repeat expansion in the first exon of the disease gene, HD. CAG DNA expansion results in a polyglutamine tract expansion in mutant huntingtin protein. Wild-type and mutant full-length huntingtin have been detected in the nucleus, but elevated levels of mutant huntingtin and huntingtin amino-terminal proteolytic fragments are seen to accumulate in the nuclei of HD-affected neurons. The presence of huntingtin in both the nucleus and the cytoplasm suggested that huntingtin may be dynamic between these compartments. By live cell time-lapse video microscopy, we have been able to visualize polyglutamine-mediated aggregation and the transient nuclear localization of huntingtin over time in a striatal cell line. A classical nuclear localization signal could not be detected in huntingtin, but we have discovered a nuclear export signal (NES) in the carboxy-terminus of huntingtin. Leptomycin B treatment of clonal striatal cells enhanced the nuclear localization of huntingtin, and a mutant NES huntingtin displayed increased nuclear localization, indicating that huntingtin can shuttle to and from the nucleus. The huntingtin NES is strictly conserved among all huntingtin proteins from diverse species. This export signal may be important in Huntington's disease because this fragment of huntingtin is proteolytically cleaved away during HD. The huntingtin NES therefore defines a potential role for huntingtin as a member of a nucleocytoplasmic dynamic protein complex.

Active Transport, Cell Nucleus↗
PubMed2002

Hairpin formation in Friedreich's ataxia triplet repeat expansion.

Triplet repeat tracts occur throughout the human genome. Expansions of a (GAA)(n)/(TTC)(n) repeat tract during its transmission from parent to child are tightly associated with the occurrence of Friedreich's ataxia. Evidence supports DNA slippage during DNA replication as the cause of the expansions. DNA slippage results in single-stranded expansion intermediates. Evidence has accumulated that predicts that hairpin structures protect from DNA repair the expansion intermediates of all of the disease-associated repeats except for those of Friedreich's ataxia. How the latter repeat expansions avoid repair remains a mystery because (GAA)(n) and (TTC)(n) repeats are reported not to self-anneal. To characterize the Friedreich's ataxia intermediates, we generated massive expansions of (GAA)(n) and (TTC)(n) during DNA replication in vitro using human polymerase beta and the Klenow fragment of Escherichia coli polymerase I. Electron microscopy, endonuclease cleavage, and DNA sequencing of the expansion products demonstrate, for the first time, the occurrence of large and growing (GAA)(n) and (TTC)(n) hairpins during DNA synthesis. The results provide unifying evidence that predicts that hairpin formation during DNA synthesis mediates all of the disease-associated, triplet repeat expansions.

DNA↗
PubMed1998

Uncloned expanded CAG/CTG repeat sequences in autosomal dominant cerebellar ataxia (ADCA) detected by the repeat expansion detection (RED) method.

In some neurodegenerative diseases, genetic anticipation correlates with expansions of the CAG/CTG repeat sequence above the normal range through the generations of a pedigree. Among these neurodegenerative diseases are late onset autosomal dominant cerebellar ataxias (ADCA). ADCA are genetically heterogeneous disorders with different cloned genes for spinocerebellar ataxia type 1 (SCA1), type 2 (SCA2), type 3 or Machado-Joseph disease (SCA3/MJD), and type 6 (SCA6). Another related dominant ataxia, dentatorubral-pallidoluysian atrophy (DRPLA), also shows CAG/CTG repeat expansions. Genetic anticipation has been reported for all of them except for the recently cloned SCA6 gene. Other, as yet undetected SCA genes may show the same features. We have used the repeat expansion detection (RED) method to detect repeat expansions directly in DNA samples from ADCA patients not resulting from known genes. Our sample consists of 19 affected index cases, corresponding to 52.8% of our ADCA families without CAG/CTG repeat expansions in the SCA1, SCA2, SCA3/MJD, SCA6, or DRPLA genes. Eighty-nine percent of the index cases had expansions of a CAG/CTG sequence greater than 40 repeats by RED, while these were observed in only 26.9% of 78 healthy subjects from the general population (p < 0.0001). The distribution of RED fragments in controls and ADCA patients also shows significant differences with the Mann-Whitney U test (U = 376.5, p = 0.0007). Moreover, there was a significant inverse correlation between the size of expansion and the age of onset (r = -0.54, p = 0.018). These results show CAG/CTG repeat expansions of over 40 repeats in our sample of ADCA families not resulting from known SCA genes.

Adult↗
PubMed2000

Very late-onset Friedreich ataxia despite large GAA triplet repeat expansions.

BACKGROUND: Most patients with Friedreich ataxia (FRDA) have abnormal GAA triplet repeat expansions in both X25 genes. The size of the GAA expansion in the shorter of the 2 expanded alleles correlates significantly with parameters of clinical severity and is inversely related to the age at onset. OBJECTIVES: To describe the clinical and molecular genetic findings in a patient with very late-onset FRDA and to review the literature. PATIENT AND METHODS: A 58-year-old white woman with mild progressive gait disturbance of 15 years' duration whose examination revealed mild incoordination was analyzed for mutations in the X25 gene. A combination of long-range polymerase chain reaction and genomic Southern blot analyses were used to identify GAA expansions in intron 1 of the X25 gene. To uncover evidence of somatic variability in triplet repeat length, DNA isolated from several tissue samples was similarly analyzed. Single-strand conformational polymorphism analysis was used to screen for mutations spanning the entire coding sequence of frataxin and all intron-exon junctions of the X25 gene. RESULTS: DNA isolated from blood leukocytes revealed GAA triplet repeat expansions in both X25 genes, which were estimated to contain 835 and 1200 repeats. Similar expansions were detected in DNA isolated from lymphoblasts, fibroblasts, buccal cells, and sural nerve, with estimated mean (+/- SD) lengths of the shorter and longer expansions being 854 (+/-69) and 1283 (+/-72) triplets, respectively. A review of reported cases of late-onset Friedreich ataxia (25-39 years) and very late-onset Friedreich ataxia (> or =40 years) demonstrated that this is the first instance of a patient presenting with very late-onset FRDA despite carrying more than 800 GAA repeats in both expanded X25 alleles. CONCLUSIONS: This unique case of very late-onset FRDA highlights a limitation in our ability to accurately predict the phenotype in FRDA based solely on the size of the GAA expansion. Other genetic or environmental factors may significantly modify disease severity in FRDA.

Age of Onset↗
PubMedDate not supplied

A survey of trinucleotide/tandem repeat-containing transcripts (TNRTs) isolated from human spinal cord to identify genes containing unstable DNA regions as candidates for disorders of motor function.

Expansion of unstable DNA regions containing trinucleotide/tandem repeats (TNRs) represents a common genetic mutation in hereditary forms of neurological disorders. The spectrum of neurological diseases linked to TNR expansions has recently broadened to include conditions with both dominant and recessive inheritance and those with or without clinical anticipation. In view of the frequent involvement of the spinal cord in neurodegenerative disorders, we have analysed this key tissue to identify pathological TNRs. We have used two approaches to isolate a wide range of trinucleotide/tandem repeat-containing transcripts (TNRTs) from human spinal cord, firstly a polymerase chain reaction (PCR)-based method and secondly by screening a spinal cord cDNA library immobilised on a membrane. Overall, 97 TNRTs belonging to a number of key protein families, the most highly represented being transcription factors, intracellular signalling molecules and cytoskeletal proteins, have been isolated most of which have not previously been considered as potential disease-causing genes. The commonest repeat motifs found in our study were CAG (37%) and CCG (24%). Known genes involved in DNA repeat expansion-related neurological disorders (e.g., AAD10, Ataxin-3, Huntingtin) were detected which validated our methods. We have characterised homogeneous TNRs among the detected gene candidates in a search for potential pathological repeat expansions. The potential role of the gene candidates identified is discussed in terms of their contribution to neurodegenerative processes.

Gene Library↗
PubMed2006

Simple procedure for automatic detection of unstable alleles in the myotonic dystrophy and Huntington's disease loci.

Human neurodegenerative and neuromuscular disorders are associated with a class of gene mutations represented by expansion of trinucleotide repeats. DNA testing is important for the diagnosis of these diseases because clinical discrimination is complicated by their late onset and frequently overlapping symptomatology. However, detection of pathologic alleles expanded up to several thousand trinucleotides poses a challenge for the introduction of rapid, fully automatic, and simple DNA diagnostic procedures. Here we propose a simple two-step polymerase chain reaction (PCR) protocol for rapid molecular diagnostics of myotonic dystrophy, Huntington's disease, and possibly also other triplet expansion diseases. Standard PCR amplification with target repeat flanking primers is used for the detection of alleles of up to 100 repeats; next, triplet-primed PCR is applied for detection of larger expansions. Automated capillary electrophoresis of amplicons allows rapid discrimination between normal, premutated and expanded (CTG/CAG)(n) alleles. Using the suggested protocol, the expanded allele was successfully detected in all test DNA samples with known genotypes. Our experience demonstrates that the suggested two-step PCR protocol provides high sensitivity, specificity, and reproducibility; is significantly less time-consuming; is easier to perform; and provides a better basis for automation than previous methods requiring Southern analysis. Therefore, it can be used for confirmation of uncertain clinical diagnoses, for prenatal testing in at-risk families, and, generally in research on these diseases.

Alleles↗
PubMed1999

Analysis of ERDA1, CTG18.1, and uncloned CAG/CTG repeat sequences in familial Parkinson's disease with anticipation.

In several neurodegenerative diseases, anticipation or increase in disease severity in succeeding generations within families correlates with expansions of an intragenic CAG/CTG repeat sequence above the normal range through the generations of a pedigree. Some kindreds of familial Parkinson's disease (PD) exhibit genetic anticipation. We used the repeat expansion detection (RED) method to detect repeat expansions directly in DNA samples from the index cases of 34 different PD families with anticipation. The mean age at onset of the younger probands was 48.8 +/- 10.8 years and the mean intergenerational difference was 19.2 +/- 10 years. The distribution of the RED products greater than 40 repeats was not significantly different between patients and controls with the Mann-Whitney U test (U = 510.5, p = 0.67). The samples were then screened for the two expanded-repeat loci, ERDA1 and CTG18.1. We found that in all cases the repeat expansion detected by the RED method may be accounted for by an expansion at these loci. Our results demonstrate that unstable CAG/CTG expansions corresponding to uncloned or cloned sequences (ERDA1, CTG18.1) are not involved in the etiology of rare familial case of PD with genetic anticipation. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 88:738-741, 1999

Adult↗