Irina V. Kovtun

OGG1 initiates age-dependent CAG trinucleotide expansion in somatic cells

Although oxidative damage has long been associated with ageing and neurological disease, mechanistic connections of oxidation to these phenotypes have remained elusive. Here we show that the age-dependent somatic mutation associated with Huntington’s disease occurs in the process of removing oxidized base lesions, and is remarkably dependent on a single base excision repair enzyme, 7,8-dihydro-8-oxoguanine-DNA glycosylase (OGG1). Both in vivo and in vitro results support a ‘toxic oxidation’ model in which OGG1 initiates an escalating oxidation–excision cycle that leads to progressive age-dependent expansion. Age-dependent CAG expansion provides a direct molecular link between oxidative damage and toxicity in post-mitotic neurons through a DNA damage response, and error-prone repair of single-strand breaks.

Trinucleotide expansion in haploid germ cells by gap repair

Huntington disease (HD) is one of eight progressive neurodegenerative disorders in which the underlying mutation is a CAG expansion encoding a polyglutamine tract. The mechanism of trinucleotide expansion is poorly understood. Expansion is mediated by misaligned pairing of repeats and the inappropriate formation of DNA secondary structure as the duplex unpairs. It has never been clear, however, whether duplex unpairing occurs during mitotic replication or during strand-break repair. In simple organisms, trinucleotide expansion arises by replication slippage on either the leading or the lagging strand, homologous recombination, gene conversion, double-strand break repair and base excision repair; it is not clear which of these mechanisms is used in mammalian cells in vivo. We have followed heritable changes in CAG length in male transgenic mice. In germ cells, expansion is limited to the post-meiotic, haploid cell and therefore cannot involve mitotic replication or recombination between a homologous chromosome or a sister chromatid. Our data support a model in which expansion in the germ cells arises by gap repair and depends on a complex containing Msh2. Expansion occurs during gap-filling synthesis when DNA loops comprising the CAG trinucleotide repeats are sealed into the DNA strand.

Genome-wide analysis reveals recurrent structural abnormalities of TP63 and other p53-related genes in peripheral T-cell lymphomas

Peripheral T-cell lymphomas (PTCLs) are aggressive malignancies of mature T lymphocytes with 5-year overall survival rates of only ∼ 35%. Improvement in outcomes has been stymied by poor understanding of the genetics and molecular pathogenesis of PTCL, with a resulting paucity of molecular targets for therapy. We developed bioinformatic tools to identify chromosomal rearrangements using genome-wide, next-generation sequencing analysis of mate-pair DNA libraries and applied these tools to 16 PTCL patient tissue samples and 6 PTCL cell lines. Thirteen recurrent abnormalities were identified, of which 5 involved p53-related genes (TP53, TP63, CDKN2A, WWOX, and ANKRD11). Among these abnormalities were novel TP63 rearrangements encoding fusion proteins homologous to ΔNp63, a dominant-negative p63 isoform that inhibits the p53 pathway. TP63 rearrangements were seen in 11 (5.8%) of 190 PTCLs and were associated with inferior overall survival; they also were detected in 2 (1.2%) of 164 diffuse large B-cell lymphomas. As TP53 mutations are rare in PTCL compared with other malignancies, our findings suggest that a constellation of alternate genetic abnormalities may contribute to disruption of p53-associated tumor suppressor function in PTCL.

Features of trinucleotide repeat instability in vivo

Unstable repeats are associated with various types of cancer and have been implicated in more than 40 neurodegenerative 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.

Microtubule destabilization and nuclear entry are sequential steps leading to toxicity in Huntington's disease

There has been a longstanding debate regarding the role of proteolysis in Huntingtons disease. The toxic peptide theory posits that N-terminal cleavage fragments of mutant Huntingtons disease protein [mutant huntingtin (mhtt)] enter the nucleus to cause transcriptional dysfunction. However, recent data suggest a second model in which proteolysis of full-length mhtt is inhibited. Importantly, the two competing theories differ with respect to subcellular distribution of mhtt at initiation of toxicity: nuclear if cleaved and cytoplasmic in the absence of cleavage. Using quantitative single-cell analysis and time-lapse imaging, we show here that transcriptional dysfunction is “downstream” of cytoplasmic dysfunction. Primary and reversible toxic events involve destabilization of microtubules mediated by full-length mhtt before cleavage. Restoration of microtubule structure by taxol inhibits nuclear entry and increases cell survival.

Gene Panel Model Predictive of Outcome in Men at High-Risk of Systemic Progression and Death From Prostate Cancer After Radical Retropubic Prostatectomy

PURPOSE In men who are at high-risk of prostate cancer, progression and death from cancer after radical retropubic prostatectomy (RRP), limited prognostic information is provided by established prognostic features. The objective of this study was to develop a model predictive of outcome in this group of patients. METHODS Candidate genes were identified from microarray expression data from 102 laser capture microdissected prostate tissue samples. Candidates were overexpressed in tumor compared with normal prostate and more frequently in Gleason patterns 4 and 5 than in 3. A case control study of 157 high-risk patients, matched on Gleason score and stage with systemic progression or death of prostate cancer as the end point, was used to evaluate the expression of candidate genes and build a multivariate model. Tumor was collected from the highest Gleason score in paraffin-embedded blocks and the gene expression was quantified by real-time reverse transcription polymerase chain reaction. Validation of the final model was performed on a separate case-control study of 57 high-risk patients who underwent RRP. RESULTS A model incorporating gene expression of topoisomerase-2a, cadherin-10, the fusion status based on ERG, ETV1, and ETV4 expression, and the aneuploidy status resulted in a 0.81 area under the curve (AUC) in receiver operating characteristic statistical analysis for the identification of men with systemic progression and death from high grade prostate cancer. The AUC was 0.79 in the independent validation study. CONCLUSION The model can identify men with high-risk prostate cancer who may benefit from more intensive postoperative follow-up and adjuvant therapies.

Lineage Relationship of Gleason Patterns in Gleason Score 7 Prostate Cancer

Gleason score 7 (GS7) prostate cancer [tumors with both Gleason patterns 3 (GP3) and 4 (GP4)] portends a significantly more aggressive tumor than Gleason score 6 (GS6). It is, therefore, critical to understand the molecular relationship of adjacent GP3 and GP4 tumor cell populations and relate molecular abnormalities to disease progression. To decipher molecular relatedness, we used laser capture microdissection (LCM) and whole-genome amplification (WGA) to separately collect and amplify DNA from adjacent GP3 and GP4 cell populations from 14 cases of GS7 prostate cancer. We then carried out massively parallel mate-pair next generation sequencing (NGS) to examine the landscape of large chromosomal alterations. We identified four to 115 DNA breakpoints in GP3 and 17 to 480 in GP4. Our findings indicate that while GP3 and GP4 from the same tumor each possess unique breakpoints, they also share identical ones, indicating a common origin. Approximately 300 chromosomal breakpoints were localized to the regions affected in at least two tumors, whereas more than 3,000 were unique within the set of 14 tumors. TMPRSS2-ERG was the most recurrent rearrangement present in eight cases, in both GP3 and GP4. PTEN rearrangements were found in five of eight TMPRSS2-ERG fusion-positive cases in both GP3 and GP4. Hierarchical clustering analysis revealed that GP3 has greater breakpoint similarity to its partner GP4 compared with GP3 from different patients. We show evidence that LCM, WGA, and NGS of adjacent tumor regions provide an important tool in deciphering lineage relationships and discovering chromosomal alterations associated with tumor progression.

Quantification of Somatic Chromosomal Rearrangements in Circulating Cell-Free DNA from Ovarian Cancers.

Recently, the use of a liquid biopsy has shown promise in monitoring tumor burden. While point mutations have been extensively studied, chromosomal rearrangements have demonstrated greater tumor specificity. Such rearrangements can be identified in the tumor and subsequently detected in the plasma of patients using quantitative PCR (qPCR). In this study we used a whole-genome mate-pair protocol to characterize a landscape of genomic rearrangements in the primary tumors of ten ovarian cancer patients. Individualized tumor-specific primer panels of aberrant chromosomal junctions were identified for each case and detected by qPCR within the cell-free DNA. Selected chromosomal junctions were detected in pre-surgically drawn blood in eight of the ten patients. Of these eight, three demonstrated the continued presence of circulating tumor DNA (ctDNA) post-surgery, consistent with their documented presence of disease, and in five ctDNA was undetectable in the post-surgical blood collection, consistent with their lack of detectable disease. The ctDNA fraction was calculated using a novel algorithm designed for the unique challenges of quantifying ctDNA using qPCR to allow observations of real-time tumor dynamics. In summary, a panel of individualized junctions derived from tumor DNA could be an effective way to monitor cancer patients for relapse and therapeutic efficacy using cfDNA.

Triplet Repeats and DNA Repair

This chapter describes methods for the isolation of specific cell types that reveal how and where expansion can occur. For the hereditary component of expansion, the male germ cell has proved useful in distinguishing processes that can contribute to expansion, as described in our article (Nature Genetics 27, 407, 2001). Mature spermatazoa (SZs) can be isolated directly from the epididymis. Haploid spermatids (STs), diploid spermatagonia (SGs), and tetraploid spermatocytes (SCs) can be removed from the testis and sorted by fluorescence-activity cell sorting (FACS); differences in DNA content and morphology allow resolution by fluorescence and light scattering. Repeat-length measurement can pinpoint the stage at which expansion occurs. Because the timing of meiosis and mitosis with respect to sperm development is known, the analysis can distinguish repair and replication processes. Furthermore, the possible contribution of Y- or X-specific factors can be evaluated by sorting X- and Y-bearing germ cells. To enable analysis of female germ cells, we describe methods for oocyte preparations and a method for the isolation of the eight-cell-stage embryo. Therefore, the methods described here can help to answer such questions as the timing during development of expansion, whether expansion is limited to a single period, whether both replication and repair contribute to instability, and the role of somatic instability in disease. If further expansion of the inherited allele contributes to the phenotype, then intervention in somatic tissue might be therapeutic.

Chromosomal catastrophe is a frequent event in clinically insignificant prostate cancer

Massive genomic rearrangements, a result of single catastrophic event termed chromothrispsis or chromosomal catastrophe, have been identified in a variety of human cancers. In a few cancer types, chromothripsis was found to be associated with poor prognosis. We performed mate-pair sequencing and analysis of structural rearrangements in 132 prostate cancer cases which included clinically insignificant Gleason score 6 tumors, clinically significant tumors of higher grade (7+) and high grade prostatic intraepithelial neoplasia. Chromothripsis was observed at least 30 per cent of the samples across different grades. Surprisingly, it was frequently observed in clinically insignificant Gleason score 6 tumors, indicating that chromothripsis does not define more aggressive phenotype. The degree of chromothripsis did not increase significantly in tumors of advanced grades and did not appear to contribute to tumor progression. Our data showed that distribution of chromothriptic rearrangements differed from that of fragile sites but correlated with the size of chromosomes. We also provided evidence that rearrangements resulting from chromothripsis were present in the cells of neighboring Gleason patterns of the same tumor. Our data suggest that that chromothripsis plays role in prostate cancer initiation.