Psychosis and genes with trinucleotide repeat polymorphism

Abnormal expansion of genes with trinucleotide repeat (TNR) polymorphism has been found in a number of neuropsychiatric disorders. These disorders and the major psychoses, schizophrenia and bipolar affective disorder, appear to share an interesting phenomenon: genetic anticipation. Because TNR expansion correlates with anticipation, these unstable DNA sites are considered important candidate loci for the major psychoses. We investigated genes with TNR polymorphisms, includingB1, B33, B37, and theN-cadherin gene, in unrelated Caucasian North American and Italian schizophrenics (n = 53 to 74), and matched controls. Also, unrelated Caucasian North American patients with bipolar I affective disorder were screened for the B33 andN-cadherin genes (n = 49 and 63, respectively). No unusually long alleles that would suggest abnormal expansion of the TNR were observed for any of these genes. Also, no statistically significant results were found in tests for genetic association between any of these genes and schizophrenia. For B37, a trend toward a difference in allele counts between schizophrenics and controls was observed. However, no clear evidence for a role of these TNR-containing genes in schizophrenia or bipolar affective disorders was found.     

Mechanisms of trinucleotide repeat instability during human development

Trinucleotide expansion underlies several human diseases. Expansion occurs during multiple stages of human development in different cell types, and is sensitive to the gender of the parent who transmits the repeats. Repair and replication models for expansions have been described, but we do not know whether the pathway involved is the same under all conditions and for all repeat tract lengths, which differ among diseases. Currently, researchers rely on bacteria, yeast and mice to study expansion, but these models differ substantially from humans. We need now to connect the dots among human genetics, pathway biochemistry and the appropriate model systems to understand the mechanism of expansion as it occurs in human disease.     

Mitochondrial DNA mutations in human diseases: a review.

Human mitochondrial diseases have been associated recently with mitochondrial DNA mutations, duplications and deletions which impair the protein synthesis of the mitochondrial subunits of the respiratory chain complexes. A constant feature is the coincident presence of the mutated and wild type genomes which provide heteroplasmy. The clinical expression of these diseases depends on the relative expression of each kind of mitochondrial DNA in the various tissues, which in turn affects the production of ATP in these tissues. Research on nuclear gene products interfering with mtDNA or with its gene products is the next step towards understanding the etiology of these diseases.     

Detection of radiation and cyclophosphamide-induced mutations in individual mouse sperm at a human expanded trinucleotide repeat locus transgene

A method to measure the germline mutations induced by cancer treatment in humans is needed. To establish such a method we used a transgenic mouse model consisting of a human DNA repeat locus that has a high spontaneous mutation frequency as a biomarker. Alterations in repeat number were measured in individual sperm from mice hemizygous for an expanded (CTG)(162) human myotonic dystrophy type 1 (DM1) microsatellite repeat using single genome-equivalent (g.e.) PCR and detection by a DNA fragment analyzer. Mutation frequencies were measured in DNA from sperm from controls and sperm derived from stem spermatogonia, differentiating spermatogonia, and spermatocytes exposed to radiation and from spermatocytes of mice treated with cyclophosphamide. There was no increase above control levels in mutations, scored as >1 repeat changes, in any of the treated groups. However, moderately large deletion mutants (between 9 and 20 repeat changes) were observed at frequencies of 2.2% when spermatocytes were treated with cyclophosphamide and, 1.8 and 2.5% when spermatocytes and stem cells, respectively, were treated with radiation, which were significantly higher than the frequency of 0.3% in controls. Thus, radiation and cyclophosphamide induced deletions in the expanded DM1 trinucleotide repeat. PCR artifacts were characterized in sperm DNA from controls and from mice treated with radiation; all artifacts involved losses of more than 20 DM1 repeats, and surprisingly the artifact frequency was higher in treated sperm than in control sperm. The radiation-induced increase in the frequency of PCR artifacts might reflect alterations in sperm DNA that destabilize the genome not only during PCR amplification but also during early embryonic development.     

Features of trinucleotide repeat instability in vivo

Unstable repeats are associated with various types of cancer and have been implicated in more than 40 neurode-generative disorders. Trinucleotide repeats are located in non-coding and coding regions of the genome. Studies of bacteria, yeast, mice and man have helped to unravel some features of the mechanism of trinucleotide expansion. Looped DNA structures comprising trinucleotide repeats are processed during replication and/or repair to generate deletions or expansions. Most in vivo data are consistent with a model in which expansion and deletion occur by different mechanisms. In mammals, microsatellite instability is complex and appears to be influenced by genetic, epigenetic and developmental factors.     

DNA mismatch repair in trinucleotide repeat instability

Trinucleotide repeat expansions cause over 30 severe neuromuscular and neurodegenerative disorders, including Huntington's disease, myotonic dystrophy type 1, and fragile X syndrome. Although previous studies have substantially advanced the understanding of the disease biology, many key features remain unknown. DNA mismatch repair(MMR) plays a critical role in genome maintenance by removing DNA mismatches generated during DNA replication. However, MMR components,particularly mismatch recognition protein MutSβ and its interacting factors MutLα and MutLγ, have been implicated in trinucleotide repeat instability. In this review, we will discuss the roles of these key MMR proteins in promoting trinucleotide repeat instability.     

A novel approach to investigate tissue-specific trinucleotide repeat instability

Background

In Huntington's disease (HD), an expanded CAG repeat produces characteristic striatal neurodegeneration. Interestingly, the HD CAG repeat, whose length determines age at onset, undergoes tissue-specific somatic instability, predominant in the striatum, suggesting that tissue-specific CAG length changes could modify the disease process. Therefore, understanding the mechanisms underlying the tissue specificity of somatic instability may provide novel routes to therapies. However progress in this area has been hampered by the lack of sensitive high-throughput instability quantification methods and global approaches to identify the underlying factors.

Results

Here we describe a novel approach to gain insight into the factors responsible for the tissue specificity of somatic instability. Using accurate genetic knock-in mouse models of HD, we developed a reliable, high-throughput method to quantify tissue HD CAG repeat instability and integrated this with genome-wide bioinformatic approaches. Using tissue instability quantified in 16 tissues as a phenotype and tissue microarray gene expression as a predictor, we built a mathematical model and identified a gene expression signature that accurately predicted tissue instability. Using the predictive ability of this signature we found that somatic instability was not a consequence of pathogenesis. In support of this, genetic crosses with models of accelerated neuropathology failed to induce somatic instability. In addition, we searched for genes and pathways that correlated with tissue instability. We found that expression levels of DNA repair genes did not explain the tissue specificity of somatic instability. Instead, our data implicate other pathways, particularly cell cycle, metabolism and neurotransmitter pathways, acting in combination to generate tissue-specific patterns of instability.

Conclusion

Our study clearly demonstrates that multiple tissue factors reflect the level of somatic instability in different tissues. In addition, our quantitative, genome-wide approach is readily applicable to high-throughput assays and opens the door to widespread applications with the potential to accelerate the discovery of drugs that alter tissue instability.     

A small unstructured nucleic acid disrupts a trinucleotide repeat hairpin

A variety of neurodegenerative disorders are associated with the expansion of trinucleotide repeat (TNR) sequences. These repetitive sequences are prone to adopting non-canonical structures, such as intrastrand stem-loop hairpins. Indeed, the formation and persistence of these hairpins during DNA replication and/or repair have been proposed as factors that facilitate TNR expansion. Given this proposed contribution of TNR hairpins to the expansion mechanism, disruption of such structures via strand invasion offers a potential means to negate the disease-initiating expansion. In this work, we investigated the strand invading abilities of a (CTG)₃ unstructured nucleic acid on a (CAG)₁₀ TNR hairpin. Using fluorescence, optical, and electrophoretic methods, instantaneous disruption of the (CAG)₁₀ hairpin by (CTG)₃ was observed at low temperatures. Additionally, we have identified three distinct duplex-like species that form between (CAG)₁₀ and (CTG)₃; these include 1, 2, or 3 (CTG)₃ sequences hybridized to (CAG)₁₀. The results presented here showcase (CTG)₃ as an invader of a TNR hairpin and suggest that unstructured nucleic acids could serve as a scaffold to design agents to prevent TNR expansion.     

Cis-elements governing trinucleotide repeat instability in Saccharomyces cerevisiae

Trinucleotide repeat (TNR) instability in humans is governed by unique cis-elements. One element is a threshold, or minimal repeat length, conferring frequent mutations. Since thresholds have not been directly demonstrated in model systems, their molecular nature remains uncertain. Another element is sequence specificity. Unstable TNR sequences are almost always CNG, whose hairpin-forming ability is thought to promote instability by inhibiting DNA repair. To understand these cis-elements further, TNR expansions and contractions were monitored by yeast genetic assays. A threshold of approximately 15--17 repeats was observed for CTG expansions and contractions, indicating that thresholds function in organisms besides humans. Mutants lacking the flap endonuclease Rad27p showed little change in the expansion threshold, suggesting that this element is not altered by the presence or absence of flap processing. CNG or GNC sequences yielded frequent mutations, whereas A-T rich sequences were substantially more stable. This sequence analysis further supports a hairpin-mediated mechanism of TNR instability. Expansions and contractions occurred at comparable rates for CTG tract lengths between 15 and 25 repeats, indicating that expansions can comprise a significant fraction of mutations in yeast. These results indicate that several unique cis-elements of human TNR instability are functional in yeast.     

iTIS-PseTNC: A sequence-based predictor for identifying translation initiation site in human genes using pseudo trinucleotide composition

Translation is a key process for gene expression. Timely identification of the translation initiation site (TIS) is very important for conducting in-depth genome analysis. With the avalanche of genome sequences generated in the postgenomic age, it is highly desirable to develop automated methods for rapidly and effectively identifying TIS. Although some computational methods were proposed in this regard, none of them considered the global or long-range sequence-order effects of DNA, and hence their prediction quality was limited. To count this kind of effects, a new predictor, called “iTIS-PseTNC,” was developed by incorporating the physicochemical properties into the pseudo trinucleotide composition, quite similar to the PseAAC (pseudo amino acid composition) approach widely used in computational proteomics. It was observed by the rigorous cross-validation test on the benchmark dataset that the overall success rate achieved by the new predictor in identifying TIS locations was over 97%. As a web server, iTIS-PseTNC is freely accessible at http://lin.uestc.edu.cn/server/iTIS-PseTNC. To maximize the convenience of the vast majority of experimental scientists, a step-by-step guide is provided on how to use the web server to obtain the desired results without the need to go through detailed mathematical equations, which are presented in this paper just for the integrity of the new prection method.     

DNA base excision repair: a mechanism of trinucleotide repeat expansion

The expansion of trinucleotide repeat (TNR) sequences in human DNA is considered to be a key factor in the pathogenesis of more than 40 neurodegenerative diseases. TNR expansion occurs during DNA replication and also, as suggested by recent studies, during the repair of DNA lesions produced by oxidative stress. In particular, the oxidized guanine base 8-oxoguanine within sequences containing CAG repeats may induce formation of pro-expansion intermediates through strand slippage during DNA base excision repair (BER). In this article, we describe how oxidized DNA lesions are repaired by BER and discuss the importance of the coordinated activities of the key repair enzymes, such as DNA polymerase β, flap endonuclease 1 (FEN1) and DNA ligase, in preventing strand slippage and TNR expansion.     

A universal mechanism ties genotype to phenotype in trinucleotide diseases

Trinucleotide hereditary diseases such as Huntington disease and Friedreich ataxia are cureless diseases associated with inheriting an abnormally large number of DNA trinucleotide repeats in a gene. The genes associated with different diseases are unrelated and harbor a trinucleotide repeat in different functional regions; therefore, it is striking that many of these diseases have similar correlations between their genotype, namely the number of inherited repeats and age of onset and progression phenotype. These correlations remain unexplained despite more than a decade of research. Although mechanisms have been proposed for several trinucleotide diseases, none of the proposals, being disease-specific, can account for the commonalities among these diseases. Here, we propose a universal mechanism in which length-dependent somatic repeat expansion occurs during the patient's lifetime toward a pathological threshold. Our mechanism uniformly explains for the first time to our knowledge the genotype–phenotype correlations common to trinucleotide disease and is well-supported by both experimental and clinical data. In addition, mathematical analysis of the mechanism provides simple explanations to a wide range of phenomena such as the exponential decrease of the age-of-onset curve, similar onset but faster progression in patients with Huntington disease with homozygous versus heterozygous mutation, and correlation of age of onset with length of the short allele but not with the long allele in Friedreich ataxia. If our proposed universal mechanism proves to be the core component of the actual mechanisms of specific trinucleotide diseases, it would open the search for a uniform treatment for all these diseases, possibly by delaying the somatic expansion process.