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Inhibition of DNA replication by a d(CAG) repeat binding ligand.

Trinucleotide repeat expansions in genomic DNA are the molecular basis of a number of genetic diseases. The (CAG)n, (CTG)n, and (CGG)n repeats share the common sequence CXG, and inherently produce a hairpin structure involving X-X mismatch base pairs flanked by two G-C base pairs. The chance of a strand slippage leading to the repeat expansion is considered to increase with increasing stability of the hairpin form. Here we show our synthetic ligand naphthyridine-azaquinolone (NA) stabilized a hairpin form of the (CAG)n repeat and inhibited the polymerase-mediated DNA synthesis.

Adenine↗

Muscleblind localizes to nuclear foci of aberrant RNA in myotonic dystrophy types 1 and 2.

The phenotypes in myotonic dystrophy types 1 and 2 (DM1 and DM2) are similar, suggesting a shared pathophysiologic mechanism. DM1 is caused by expansion of a CTG repeat in the DMPK gene. Pathogenic effects of this mutation are likely to be mediated, at least in part, by the expanded CUG repeat in mutant mRNA. The mutant transcripts are retained in the nucleus in multiple discrete foci. We investigated the possibility that DM2 is also caused by expansion of a CTG repeat or related sequence. Analysis of DNA by repeat expansion detection methods, and RNA by ribonuclease protection, did not show an expanded CTG or CUG repeat in DM2. However, hybridization of muscle sections with fluorescence-labeled CAG-repeat oligonucleotides showed nuclear foci in DM2 similar to those seen in DM1. Nuclear foci were present in all patients with symptomatic DM1 (n = 9) or DM2 (n = 9) but not in any disease controls or healthy subjects (n = 23). The foci were not seen with CUG- or GUC-repeat probes. Foci in DM2 were distinguished from DM1 by lower stability of the probe-target duplex, suggesting that a sequence related to the DM1 CUG expansion accumulates in the DM2 nucleus. Muscleblind proteins, which interact with expanded CUG repeats in vitro, localized to the nuclear foci in both DM1 and DM2. These results support the idea that nuclear accumulation of mutant RNA is pathogenic in DM1, suggest that a similar disease process occurs in DM2, and point to a role for muscleblind in the pathogenesis of both disorders.

Adult↗

Hairpin formation during DNA synthesis primer realignment in vitro in triplet repeat sequences from human hereditary disease genes.

Genetic expansion of DNA triplet repeat sequences (TRS) found in neurogenetic disorders may be due to abnormal DNA replication. We have previously observed strong DNA synthesis pausings at specific loci within the long tracts (> approximately 70 repeats) of CTG.CAG and CGG.CCG as well as GTC.GAC by primer extensions in vitro using DNA polymerases (the Klenow fragment of Escherichia coli DNA polymerase I, the modified T7 DNA polymerase (Sequenase), and human DNA polymerase beta). Herein, we have isolated and analyzed the products of stalled synthesis found at approximately 30-40 triplets from the beginning of the TRS. DNA sequence analyses revealed that the stalled products contained short tracts of homogeneous TRS (6-12 repeats) in the middle of the sequence corresponding to the flanking region of the primer-template sequence. The sequence at the 3'-side terminated at the end of the primer, indicating that the primer molecule had served as a template. In addition, chemical probe and polyacrylamide gel electrophoretic analyses revealed that the stalled products existed in hairpin structures. We postulate that these products of the DNA polymerases are caused by the existence of an unusual DNA conformation(s) within the TRS, during the in vitro DNA synthesis, enhancing the DNA slippages and the hairpin formations in the TRS due to primer realignment. The consequence of these steps is DNA synthesis to the end of the primer and termination. Primer realignment including hairpin formation may play an important intermediate role in the replication of TRS in vivo to elicit genetic expansions.

Betaine↗

A molecular pathogenesis for transcription factor associated poly-alanine tract expansions.

Poly-alanine (Ala) tract expansions in transcription factors have been shown to be associated with human birth defects such as malformations of the brain, the digits, and other structures. Expansions of a poly-Ala tract from 15 to 22 (+7)-29 (+14) Ala in Hoxd13, for example, result in the limb malformation synpolydactyly in humans and in mice [synpolydactyly homolog (spdh)]. Here, we show that an increase of the Ala repeat above a certain length (22 Ala) is associated with a shift in the localization of Hoxd13 from nuclear to cytoplasmic, where it forms large amorphous aggregates. We observed similar aggregates for expansion mutations in SOX3, RUNX2 and HOXA13, pointing to a common mechanism. Cytoplasmic aggregation of mutant Hoxd13 protein is influenced by the length of the repeat, the level of expression and the efficacy of degradation by the proteasome. Heat shock proteins Hsp70 and Hsp40 co-localize with the aggregates and activation of the chaperone system by geldanamycin leads to a reduction of aggregate formation. Furthermore, recombinant mutant Hoxd13 protein forms aggregates in vitro demonstrating spontaneous misfolding of the protein. We analyzed the mouse mutant spdh, which harbors a +7 Ala expansion in Hoxd13 similar to the human synpolydactyly mutations, as an in vivo model and were able to show a reduction of mutant Hoxd13 and, in contrast to wt Hoxd13, a primarily cytoplasmic localization of the protein. Our results provide evidence that poly-Ala repeat expansions in transcription factors result in misfolding, degradation and cytoplasmic aggregation of the mutant proteins.

Animals↗

Post-zygotic de novo trinucleotide repeat expansion at spinocerebellar ataxia type 7 locus: evidence from an Indian family.

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant cerebellar ataxia caused by CAG repeat expansion. We found expansion at SCA7 locus in only two out of 235 Indian families clinically diagnosed for ataxia. In one of the families, a de novo mutation was observed wherein a paternal allele in intermediate range of 31 CAG repeats expanded to 59 in the offspring leading to the disease. No expanded alleles were observed in the sperm of the transmitting parent by small pool PCR. This suggests that de novo expansion by a pre-zygotic event is unlikely and could be post-zygotic. SCA7 expanded alleles from the two families were present on different genetic backgrounds, indicating multiple origins of the mutation.

DNA Repeat Expansion↗

Gene repeat expansion and contraction by spontaneous intrachromosomal homologous recombination in mammalian cells.

Homologous recombination (HR) is important in repairing errors of replication and other forms of DNA damage. In mammalian cells, potential templates include the homologous chromosome, and after DNA replication, the sister chromatid. Previous work has shown that the mammalian recombination machinery is organized to suppress interchromosomal recombination while preserving intrachromosomal HR. In the present study, we investigated spontaneous intrachromosomal HR in mouse hybridoma cell lines in which variously numbered tandem repeats of the mu heavy chain constant (C mu) region reside at the haploid, chromosomal immunoglobulin mu heavy chain locus. This organization provides the opportunity to investigate recombination between homologous gene repeats in a well-defined chromosomal locus under conditions in which recombinants are conveniently recovered. This system revealed several features about the mammalian intrachromosomal HR process: (i) the frequency of HR was high (recombinants represented as much as several percent of the total of recombinants and non-recombinants); (ii) the recombination process appeared to be predominantly non-reciprocal, consistent with the possibility of gene conversion; (iii) putative gene conversion tracts were long (up to 13.4 kb); (iv) the recombination process occurred with precision, initiating and terminating within regions of shared homology. The results are discussed with respect to mammalian intrachromosomal HR involving interactions both within and between sister chromatids.

Animals↗

The hereditary spinocerebellar ataxias in Japan.

In Japan, multiple system atrophy (MSA) accounts for 40% of all spinocerebellar ataxias (SCAs) and hereditary disorders account for 30%. Among the latter, autosomal dominant disorders are common and recessive ataxias are rare. Although the frequency of SCA genotypes differs between geographic regions throughout Japan, SCA6, SCA3/MJD, and DRPLA are the three major disorders, while SCA7, SCA8, SCA10, SCA12, and SCA17 are infrequent or almost undetected. SCA1 predominantly occurs in the northern part of Japan. Overall, 20-40% of dominant SCAs are due to unknown mutations. From this cluster, pure cerebellar ataxias linked with the SCA4, SCA14, and SCA16 locus have been isolated. Among the recessive SCAs, patients with AVED and EAOH have been detected. However, FRDA associated with GAA repeat expansion in the frataxin gene has not been reported so far.

DNA Repeat Expansion↗

Genetics and molecular pathogenesis of the myotonic dystrophies.

Pathogenic repeat expansions were initially identified as causing either a loss of gene product, such as in fragile X mental retardation, or an expansion of a polyglutamine region of a protein, as was first shown in spinobulbar muscular atrophy (Kennedy's disease). The pathogenic effect of the repeat expansion in myotonic dystrophy type 1, however, has been controversial because it does not encode a protein but nonetheless results in a highly penetrant dominant disease. Clinical and molecular characterization of myotonic dystrophy types 1 and 2 have now demonstrated a novel disease mechanism involving pathogenic effects of repeat expansions that are expressed in RNA but are not translated into protein.

DNA Repeat Expansion↗

Prevalence of FMR1 repeat expansions in movement disorders. A systematic review.

We reviewed prevalence rates of fragile X mental retardation gene (FMR1) repeat expansions in movement disorder populations. Inclusion criteria included published epidemiological studies from systematic searches of Medline, Pubmed, Cochrane Databases and Web Science. Thirteen cross-sectional studies were carried out between 2003 and 2005. Subjects with ataxia showed higher than expected rates while those with essential tremor and parkinsonism showed lower rates. The heterogeneous design of the studies, inclusion criteria and mean age of subjects may have led to underestimation of FMR1 repeat expansion prevalence rates.

Cross-Sectional Studies↗

Interruptions in the expanded ATTCT repeat of spinocerebellar ataxia type 10: repeat purity as a disease modifier?

Spinocerebellar ataxia type 10 (SCA10) is one of numerous genetic disorders that result from simple repeat expansions. SCA10 is caused by expansion of an intronic ATTCT pentanucleotide repeat tract. It is clinically characterized by progressive ataxia, seizures, and anticipation, which can vary within and between families. We report two SCA10 families showing distinct frequencies of seizures and correlations of repeat length with age at onset. One family displayed uninterrupted ATTCT expansions, whereas the other showed multiple interruptions of the repeat by nonconsensus repeat units, which differed both in the length and/or sequence of the repeat unit. Disease-causing microsatellite expansions have been assumed to be composed of uninterrupted pure repeats. Our findings for SCA10 challenge this convention and suggest that the purity of the expanded repeat element may be a disease modifier.

Age of Onset↗

Genetic basis of schizophrenia: trinucleotide repeats. An update.

1. Recent developments in technologies permit systematic screening of the entire human genome as a strategy for identification of susceptibility genes of small effect that influence risk to complex traits, like schizophrenia (Schz), inflammatory bowel disease, bipolar affective disorder (BPAD) etc. 2. Schizophrenia is known to have a high heritability and a complex inheritance pattern. Several studies provide evidence that both genes and environment play a role in the etiology of schizophrenia. Linkage studies have observed racial and sex bias in the genetic constitution of schizophrenia. Schizophrenia also manifests clinical anticipation and genomic imprinting. 3. "Dynamic mutations" or "tandem repeat expansions" in DNA, explain a number of observations associated with clinical anticipation and genomic imprinting. In patient populations, the repeat expands well beyond the normal range, altering the biological function of the gene. These sequence are unstable and increase in size between family members in successive generations, giving rise to greater severity of disease. 4. Several workers have reported an association of trinucleotide repeat length with adult- and child-onset schizophrenia. One such expanded allele has been found at the CTG18.1 locus on the 18th chromosome. Other genes known to have similar mutation are SEF2-1, which codes for a helix-loop-helix protein, hSKCa3 gene, which codes for a calcium-activated potassium channel and the transthyretin gene. In schizophrenic patients, significant difference in allele frequency distribution of these genes has been reported. 5. Population based genetic research would not only help identify different subgroups of this of schizophrenia.

Animals↗

The unstable trinucleotide repeat story of major psychosis.

New hopes for cloning susceptibility genes for schizophrenia and bipolar affective disorder followed the discovery of a novel type of DNA mutation, namely unstable DNA. One class of unstable DNA, trinucleotide repeat expansion, is the causal mutation in myotonic dystrophy, fragile X mental retardation, Huntington disease and a number of other rare Mendelian neurological disorders. This finding has led researchers in psychiatric genetics to search for unstable DNA sites as susceptibility factors for schizophrenia and bipolar affective disorder. Increased severity and decreased age at onset of disease in successive generations, known as genetic anticipation, was reported for undifferentiated psychiatric diseases and for myotonic dystrophy early in the twentieth century, but was initially dismissed as the consequence of ascertainment bias. Because unstable DNA was demonstrated to be a molecular substrate for genetic anticipation in the majority of trinucleotide repeat diseases including myotonic dystrophy, many recent studies looking for genetic anticipation have been performed for schizophrenia and bipolar affective disorder with surprisingly consistent positive results. These studies are reviewed, with particular emphasis placed on relevant sampling and statistical considerations, and concerns are raised regarding the interpretation of such studies. In parallel, molecular genetic investigations looking for evidence of trinucleotide repeat expansion in both schizophrenia and bipolar disorder are reviewed. Initial studies of genome-wide trinucleotide repeats using the repeat expansion detection technique suggested possible association of large CAG/CTG repeat tracts with schizophrenia and bipolar affective disorder. More recently, three loci have been identified that contain large, unstable CAG/CTG repeats that occur frequently in the population and seem to account for the majority of large products identified using the repeat expansion detection method. These repeats localize to an intron in transcription factor gene SEF2-1B at 18q21, a site named ERDA1 on 17q21 with no associated coding region, and the 3' end of a gene on 13q21, SCA8, that is believed to be responsible for a form of spinocerebellar ataxia. At present no strong evidence exists that large repeat alleles at either SEF2-1B or ERDA1 are involved in the etiology of schizophrenia or bipolar disorder. Preliminary evidence suggests that large repeat alleles at SCA8 that are non-penetrant for ataxia may be a susceptibility factor for major psychosis. A fourth, but much more infrequently unstable CAG/CTG repeat has been identified within the 5' untranslated region of the gene, MAB21L1, on 13q13. A fifth CAG/CTG repeat locus has been identified within the coding region of an ion transporter, KCNN3 (hSKCa3), on 1q21. Although neither large alleles nor instability have been observed at KCNN3, this repeat locus has been extensively analyzed in association and family studies of major psychosis, with conflicting findings. Studies of polyglutamine containing genes in major psychosis have also shown some intriguing results. These findings, reviewed here, suggest that, although a major role for unstable trinucleotides in psychosis is unlikely, involvement at a more modest level in a minority of cases cannot be excluded, and warrants further investigation.

Anticipation, Genetic↗

Efficient isothermal expansion of human telomeric and minisatellite repeats by Thermococcus litoralis DNA polymerase.

Repeating DNA sequences, such as telomeres, centromeres, and micro- and mini-satellites, comprise 50% of the genome and play important roles in regulatory and pathogenic mechanisms. In order to study structures and functions of such repeating sequences, it is important to have simple and efficient methods for making them in vitro. Here, we describe the efficient and convenient expansion of repetitive telomeric and minisatellite DNA sequences starting from small synthetic templates to final product lengths of several hundreds to thousands of nucleotides by the thermostable DNA polymerase from Thermococcus litoralis (Vent DNA polymerase). This enzyme was so far unknown to catalyze repeat expansion. Either single-stranded or double-stranded DNAs could be produced, depending on nucleotides present. Compared to earlier results obtained with other enzymes, the expansion reaction is highly efficient both in its yield and product length, and proceeds without thermal cycling. Moreover, the products are characterized by a narrow length distribution.

DNA↗

Flies deficient in Muscleblind protein model features of myotonic dystrophy with altered splice forms of Z-band associated transcripts.

Myotonic dystrophy (DM) is a dominantly inherited neuromuscular disorder characterised by muscle weakness and wasting. There are two forms of DM; both of which are caused by the expansion of repeated DNA sequences. DM1 is associated with a CTG repeat located in the 3' untranslated region of a gene, DMPK and DM2 with a tetranucleotide repeat expansion, CCTG, located in the first intron of a different gene, ZNF9. Recent data suggest a dominant RNA gain-of-function mechanism underlying DM, as transcripts containing either CUG or CCUG repeat expansions accumulate as foci in the nuclei of DM1 and DM2 cells respectively, where they exert a toxic effect, sequestering specific RNA binding proteins such as Muscleblind, which leads to splicing defects and the disruption of normal cellular functions. Z-band disruption is a well-known histological feature of DM1 muscle, which has also been reported in Muscleblind deficient flies. In order to determine whether there is a common molecular basis for this abnormality we have examined the alternative splicing pattern of transcripts that encode proteins associated with the Z-band in both organisms. Our results demonstrate that the missplicing of ZASP/LDB3 leads to the expression of an isoform in DM1 patient muscle, which is not present in normal controls, nor in other myopathies. Furthermore the Drosophila homologue, CG30084, is also misspliced, in Muscleblind deficient flies. Another Z-band transcript, alpha actinin, is misspliced in mbl mutant flies, but not in DM1 patient samples. These results point to similarities but subtle differences in the molecular breakdown of Z-band structures in flies and DM patients and emphasise the relevance of Muscleblind proteins in DM pathophysiology.

Actinin↗

Stability of intrastrand hairpin structures formed by the CAG/CTG class of DNA triplet repeats associated with neurological diseases.

Expansions of trinucleotide repeats in DNA, a novel source of mutations associated with human disease, may arise by DNA replication slippage initiated by hairpin folding of primer or template strands containing such repeats. To evaluate the stability of single-strand folding by repeating triplets of DNA bases, thermal melting profiles of (CAG)10, (CTG)10, (GAC)10 and (GTC)10 strands are determined at low and physiological salt concentrations, and measurements of melting temperature and enthalpy change are made in each case. Comparisons are made to strands with three times as many repeats, (CAG)30 and (CTG)30. Evidence is presented for stable intrastrand folding by the CAG/CTG class of triplet repeats. Relative to the GAC/GTC class not associated with disease, the order of folding stability is found to be CTG > GAC approximately = CAG > GTC for 10 repeats. Surprisingly, the folds formed by 30 repeats of CTG or CAG have no higher melting temperature and are only 40% more stable in free energy than those formed by 10 repeats. This finding suggests that triplet expansions with higher repeat number may result from the formation of more folded structures with similar stability rather than fewer but longer folds of greater stability.

Base Sequence↗

Pathogenic RNA repeats: an expanding role in genetic disease.

Fragile X mental retardation and Friedreich's ataxia were among the first pathogenic trinucleotide repeat disorders to be described in which noncoding repeat expansions interfere with gene expression and cause a loss of protein production. Invoking a similar loss-of-function hypothesis for the CTG expansion causing myotonic dystrophy type 1 (DM1) located in the 3' noncoding portion of a kinase gene was more difficult because DM is a dominantly inherited multisystemic disorder in which the second copy of the gene is unaffected. However, the discovery that a transcribed but untranslated CCTG expansion causes myotonic dystrophy type 2 (DM2), along with other discoveries on DM1 and DM2 pathogenesis, indicate that the CTG and CCTG expansions are pathogenic at the RNA level. This review will detail recent developments on the molecular mechanisms of RNA pathogenesis in DM, and the growing number of expansion disorders that might involve similar pathogenic RNA mechanisms.

Alternative Splicing↗

Expanded repeat in canine epilepsy.

Epilepsy afflicts 1% of humans and 5% of dogs. We report a canine epilepsy mutation and evidence for the existence of repeat-expansion disease outside humans. A canid-specific unstable dodecamer repeat in the Epm2b (Nhlrc1) gene recurrently expands, causing a fatal epilepsy and contributing to the high incidence of canine epilepsy. Tracing the repeat origins revealed two successive events, starting 50 million years ago, unique to canid evolution. A genetic test, presented here, will allow carrier and presymptomatic diagnosis and disease eradication. Clinicopathologic characterization establishes affected animals as a model for Lafora disease, the most severe teenage-onset human epilepsy.

Alleles↗