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

Effects of sequence on repeat expansion during DNA replication

Small DNA repeat tracts are located throughout the human genome. The tracts are unstable, and expansions of certain repeat sequences cause neuromuscular disease. DNA expansions appear to be associated with lagging-strand DNA synthesis and DNA repair. At some sites of repeat expansion, e.g. the myotonic dystrophy type 2 (DM2) tetranucleotide repeat expansion site, more than one repeat tract with similar sequences lie side by side. Only one of the DM2 repeat tracts, however, is found to expand. Thus, DNA base sequence is a possible factor in repeat tract expansion. Here we determined the expansion potential, during DNA replication by human DNA polymerase β, of several tetranucleotide repeat tracts in which the repeat units varied by one or more bases. The results show that subtle changes, such as switching T for C in a tetranucleotide repeat, can have dramatic consequences on the ability of the nascent-strand repeat tract to expand during DNA replication. We also determined the relative stabilities of self-annealed 100mer repeats by melting-curve analysis. The relative stabilities did not correlate with the relative potentials of the analogous repeats for expansion during DNA replication, suggesting that hairpin formation is not required for expansion during DNA replication

Data-driven asthma endotypes defined from blood biomarker and gene expression data.

The diagnosis and treatment of childhood asthma is complicated by its mechanistically distinct subtypes (endotypes) driven by genetic susceptibility and modulating environmental factors. Clinical biomarkers and blood gene expression were collected from a stratified, cross-sectional study of asthmatic and non-asthmatic children from Detroit, MI. This study describes four distinct asthma endotypes identified via a purely data-driven method. Our method was specifically designed to integrate blood gene expression and clinical biomarkers in a way that provides new mechanistic insights regarding the different asthma endotypes. For example, we describe metabolic syndrome-induced systemic inflammation as an associated factor in three of the four asthma endotypes. Context provided by the clinical biomarker data was essential in interpreting gene expression patterns and identifying putative endotypes, which emphasizes the importance of integrated approaches when studying complex disease etiologies. These synthesized patterns of gene expression and clinical markers from our research may lead to development of novel serum-based biomarker panels

Four distinct asthma endotypes identified by data-driven integration of blood gene expression and clinical biomarkers.

<p>Underlying mechanistic information is suggestive of metabolic syndrome and potential biomarkers.</p

Mechanistic interpretation of the decision tree.

<p>Cellular drivers were determined by using linear regression as described in the methods and summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117445#pone.0117445.s010" target="_blank">S1 Table</a>. The results are summarized in green boxes. Gene expression changes were interpreted using Ingenuity Pathway Analysis (IPA). The top networks from IPA are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117445#pone.0117445.s011" target="_blank">S2 Table</a> along with their significance scores. All networks that were considered as part of the functional interpretation are included as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117445#pone.0117445.s002" target="_blank">S2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117445#pone.0117445.s009" target="_blank">S9</a> Figs. The final functional summaries from this analysis are shown in blue boxes. Clinical biomarkers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117445#pone.0117445.s012" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117445#pone.0117445.s013" target="_blank">S4</a> Tables) correlated with the key genes from each metagene are shown in the purple boxes; atopy is based on allergen-specific IgE levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117445#pone.0117445.s012" target="_blank">S3 Table</a>, Phadiatop) and IgE represents total serum IgE. (A) K-PC1, no IPA network (B) B-PC2, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117445#pone.0117445.s003" target="_blank">S3 Fig</a>. (C) C-PC2, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117445#pone.0117445.s004" target="_blank">S4 Fig</a>. (D) B-PC1, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117445#pone.0117445.s002" target="_blank">S2 Fig</a>. (E) J-PC2, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117445#pone.0117445.s008" target="_blank">S8</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117445#pone.0117445.s009" target="_blank">S9</a> Figs. (F) F-PC2, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117445#pone.0117445.s007" target="_blank">S7 Fig</a>. (G) E-PC2, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117445#pone.0117445.s005" target="_blank">S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117445#pone.0117445.s006" target="_blank">S6</a> Figs.</p