Distributions of estimated relative instabilities for 50 deletion-prone cancer genes and 50 randomly chosen genes.

<p>This diagram provides separate histograms that describe the relative instabilities of the 50 deletion-prone genes and the 50 randomly selected genes, respectively. The values in these histograms are the unadjusted outputs from the <i>Alu</i> element-based instability model algorithm. These stabilities are also provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065188#pone.0065188.s003" target="_blank">Tables S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065188#pone.0065188.s004" target="_blank">Table S4</a>, respectively. Note that the least stable of all 100 genes is the randomly selected gene, <i>GDPD2</i>. This low stability is the result of the putative exonized <i>Alu</i> that occurs in variant 1 of <i>GDPD2's</i> 12^th exon (see text).</p

A Comparison of 100 Human Genes Using an <i>Alu</i> Element-Based Instability Model

<div><p>The human retrotransposon with the highest copy number is the <i>Alu</i> element. The human genome contains over one million <i>Alu</i> elements that collectively account for over ten percent of our DNA. Full-length <i>Alu</i> elements are randomly distributed throughout the genome in both forward and reverse orientations. However, full-length widely spaced <i>Alu</i> pairs having two <i>Alus</i> in the same (direct) orientation are statistically more prevalent than <i>Alu</i> pairs having two <i>Alus</i> in the opposite (inverted) orientation. The cause of this phenomenon is unknown. It has been hypothesized that this imbalance is the consequence of anomalous inverted <i>Alu</i> pair interactions. One proposed mechanism suggests that inverted <i>Alu</i> pairs can ectopically interact, exposing both ends of each <i>Alu</i> element making up the pair to a potential double-strand break, or “hit”. This hypothesized “two-hit” (two double-strand breaks) potential per <i>Alu</i> element was used to develop a model for comparing the relative instabilities of human genes. The model incorporates both 1) the two-hit double-strand break potential of <i>Alu</i> elements and 2) the probability of exon-damaging deletions extending from these double-strand breaks. This model was used to compare the relative instabilities of 50 deletion-prone cancer genes and 50 randomly selected genes from the human genome. The output of the <i>Alu</i> element-based genomic instability model developed here is shown to coincide with the observed instability of deletion-prone cancer genes. The 50 cancer genes are collectively estimated to be 58% more unstable than the randomly chosen genes using this model. Seven of the deletion-prone cancer genes, <i>ATM, BRCA1, FANCA, FANCD2, MSH2, NCOR1 and PBRM1</i>, were among the most unstable 10% of the 100 genes analyzed. This algorithm may lay the foundation for comparing genetic risks posed by structural variations that are unique to specific individuals, families and people groups.</p></div

Estimated human deletion size frequency distribution.

<p>(A) This log-log (base 10) plot estimates the relative distribution of deletion sizes within the human genome. The curve was constructed from two different studies and predicts that 95% of deletions are ≤50 bp in size and 99% of deletions are ≤445 bp <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065188#pone.0065188-Mills1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065188#pone.0065188-Wheeler1" target="_blank">[27]</a>. When combined with the two-hit hypothesis for <i>Alu</i> elements (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065188#pone-0065188-g001" target="_blank">Figure 1F</a> and text), this curve suggests that the two ends of an <i>Alu</i> element pose specific and different risks to an exon's coding region.</p

<i>Alu</i> landscapes for <i>BRCA1</i> and <i>VHL</i>.

<p>This figure characterizes the <i>Alu</i> landscapes within and 500 kbp, 5′ and 3′ of A) <i>BRCA1</i> and B) <i>VHL</i>. The midpoint for each <i>Alu</i> element is plotted against its respective instability score, <i>i</i>Score. Larger <i>i</i>Score values represent higher predicted <i>Alu</i> element instabilities (see text). Similar <i>Alu</i> landscapes for eight additional genes examined in this study are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065188#pone.0065188.s010" target="_blank">figures S4A–H</a>. These spans are twice the size of the ±250 kbp flanking landscapes which are considered to pose a risk for an exon damaging deletion (see text). These larger spans better illustrate the ebb and flow of <i>Alu</i>-related instability around each respective gene.</p

Proposed mechanism for formation and resolution of doomsday junction formed by ectopic invasion and annealing of complementary DNA breathing bubbles.

<p>(A) Two <i>Alu</i> elements in opposite orientations form an inverted <i>Alu</i> pair. (B) These inverted <i>Alu</i> pairs can align as high-homology regions. (C) DNA bubbles create short-lived sections of single-stranded DNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065188#pone.0065188-Jeon1" target="_blank">[25]</a>. (D) The unbound bases within these bubbles are characterized by their flipping out from the centerline of the DNA strand <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065188#pone.0065188-Fogedby1" target="_blank">[24]</a>. Coincident passage of these bubbles within aligned <i>Alu</i> elements can create the opportunity for interactions between the flipped-out bases of the complementary DNA strands. (E) The ectopic invasion and annealing of single-stranded DNA associated with high-homology DNA bubbles could potentially extend to the entire length of the <i>Alu</i> elements. The hypothetical conformation created by this interaction is termed a doomsday junction. A similar interaction may also occur between high-homology replication forks and is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065188#pone.0065188.s007" target="_blank">Figure S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065188#pone.0065188-Cook1" target="_blank">[18]</a>. Eight segments of single-stranded DNA formed at the boundary of doomsday junctions create the opportunity for single-strand nuclease attack. These sites are illustrated as yellow lightning bolts. (F) As again illustrated by the yellow lightning bolts, each end of each <i>Alu</i> element involved in the doomsday junction is vulnerable to a double-strand break. This two-hit hypothesis for each <i>Alu</i> element was incorporated into the model's algorithm (see text).</p

The <i>Alu</i> pair I∶D ratio versus spacer size for Type 1 <i>Alu</i> pairs for APSNs 1–10.

<p>A total of 10 points are used to construct each of the 10 APSN curves in this figure. These points represent the respective <i>Alu</i> pair I∶D ratios for 10, non-overlapping spacer size groupings. The first point (left to right) represents the I∶D ratio for the smallest five percent of spacer sizes. This point is followed by nine consecutively larger spacer size groupings. Each of these nine larger sized groupings contains 10% of the <i>Alu</i> pairs found within the respective APSN family. The I∶D ratio for each percentile group is plotted against its median spacer size, respectively (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065188#s5" target="_blank">Methods</a>). This plot illustrates that the I∶D ratio is not a continuous function versus spacer size and may indicate the activity of different <i>Alu-Alu</i> interaction mechanisms (see text). These curves, along with their 5′ mirror images, make up ten of the 220 (APSNs ±1–110) curves that are collectively shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065188#pone-0065188-g003" target="_blank">Figure 3</a>.</p

The <i>Alu</i> pair I∶D ratio versus spacer size for Type 1 <i>Alu</i> pairs for APSNs ±1–110.

<p>This figure illustrates the ±110 APSN curves for full-length (275–325 bp) Type 1 human <i>Alu</i> pairs. The individual curves in this figure are so closely spaced that they collectively appear as a surface. An expanded view of Type 1 APSN curves 1–10 is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065188#pone-0065188-g002" target="_blank">Figure 2</a>. Similar I∶D surfaces for Type 2 and Type 3 <i>Alu</i> pairs are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065188#pone.0065188.s008" target="_blank">Figures S2A and S2B</a>, respectively.</p

PCR amplification of polymorphic <i>Alu</i> insertions in Lemuriformes.

<p>Gel photographs displaying the methodology for establishing evolutionary relationships using <i>Alu</i> elements. The presence and absence of elements, supplemented by sequencing to eliminate the possibility of confounding events, is used to determine which species are more closely related. A total of 5 gel electrophoresis results on a 24-species primate panel are shown with <i>H. sapiens</i> and <i>G. senegalensis</i> as outgroups. <b>A:</b> Amplification of locus Str71B, an <i>Alu</i> insertion shared by the infraorder Lemuriformes. <b>B:</b> Amplification of locus MmA39, an <i>Alu</i> insertion shared by the family Cheirogaleidae. <b>C:</b> Amplification of locus MmA27, an <i>Alu</i> insertion shared by the sister genera <i>Microcebus</i> and <i>Mirza</i>. <b>D:</b> Amplification of locus Str59, an <i>Alu</i> insertion specific to the genus <i>Microcebus</i>. <b>E:</b> Amplification of locus Em6, an <i>Alu</i> insertion affirming the monophyly of the family Lemuridae to the exclusion of other lemur species and outgroups.</p

DNA samples of all species examined in this study.

a<p>Coriell Institute for Medical Research, 403 Haddon Avenue, Camden, NJ 08103, USA.</p>b<p>Duke Lemur Center (DLC), Duke University, Durham, NC 27708, USA.</p>c<p>Integrated Primate Biomaterials and Information Resource (IPBIR), <a href="http://ccr.coriell.org/Sections/Collections/" target="_blank">http://ccr.coriell.org/Sections/Collections/</a>.</p>d<p>Frozen Zoo, San Diego Zoo (SDFZ), <a href="http://conservationandscience.org" target="_blank">http://conservationandscience.org</a>.</p>e<p>Gene Bank of Primates (GBP), German Primate Center, Göttingen, Germany.</p>f<p>Batzer: Adenovirus 12 SV40-transformed fibroblasts maintained in the lab of Dr. Mark Batzer.</p>g<p>From cell lines provided by American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, USA.</p><p>DNA samples of all species examined in this study.</p

An <em>Alu</em>-Based Phylogeny of Lemurs (Infraorder: Lemuriformes)

<div><p>Lemurs (infraorder: Lemuriformes) are a radiation of strepsirrhine primates endemic to the island of Madagascar. As of 2012, 101 lemur species, divided among five families, have been described. Genetic and morphological evidence indicates all species are descended from a common ancestor that arrived in Madagascar ∼55–60 million years ago (mya). Phylogenetic relationships in this species-rich infraorder have been the subject of debate. Here we use <em>Alu</em> elements, a family of primate-specific Short INterspersed Elements (SINEs), to construct a phylogeny of infraorder Lemuriformes. <em>Alu</em> elements are particularly useful SINEs for the purpose of phylogeny reconstruction because they are identical by descent and confounding events between loci are easily resolved by sequencing. The genome of the grey mouse lemur (<em>Microcebus murinus</em>) was computationally assayed for synapomorphic <em>Alu</em> elements. Those that were identified as Lemuriformes-specific were analyzed against other available primate genomes for orthologous sequence in which to design primers for PCR (polymerase chain reaction) verification. A primate phylogenetic panel of 24 species, including 22 lemur species from all five families, was examined for the presence/absence of 138 <em>Alu</em> elements via PCR to establish relationships among species. Of these, 111 were phylogenetically informative. A phylogenetic tree was generated based on the results of this analysis. We demonstrate strong support for the monophyly of Lemuriformes to the exclusion of other primates, with Daubentoniidae, the aye-aye, as the basal lineage within the infraorder. Our results also suggest Lepilemuridae as a sister lineage to Cheirogaleidae, and Indriidae as sister to Lemuridae. Among the Cheirogaleidae, we show strong support for <em>Microcebus</em> and <em>Mirza</em> as sister genera, with <em>Cheirogaleus</em> the sister lineage to both. Our results also support the monophyly of the Lemuridae. Within Lemuridae we place <em>Lemur</em> and <em>Hapalemur</em> together to the exclusion of <em>Eulemur</em> and <em>Varecia</em>, with <em>Varecia</em> the sister lineage to the other three genera.</p> </div