Systematic detection of putative tumor suppressor genes through the combined use of exome and transcriptome sequencing

Abstract Background To identify potential tumor suppressor genes, genome-wide data from exome and transcriptome sequencing were combined to search for genes with loss of heterozygosity and allele-specific expression. The analysis was conducted on the breast cancer cell line HCC1954, and a lymphoblast cell line from the same individual, HCC1954BL. Results By comparing exome sequences from the two cell lines, we identified loss of heterozygosity events at 403 genes in HCC1954 and at one gene in HCC1954BL. The combination of exome and transcriptome sequence data also revealed 86 and 50 genes with allele specific expression events in HCC1954 and HCC1954BL, which comprise 5.4% and 2.6% of genes surveyed, respectively. Many of these genes identified by loss of heterozygosity and allele-specific expression are known or putative tumor suppressor genes, such as BRCA1, MSH3 and SETX, which participate in DNA repair pathways. Conclusions Our results demonstrate that the combined application of high throughput sequencing to exome and allele-specific transcriptome analysis can reveal genes with known tumor suppressor characteristics, and a shortlist of novel candidates for the study of tumor suppressor activities

Distinct patterns of somatic alterations in a lymphoblastoid and a tumor genome derived from the same individual

Although patterns of somatic alterations have been reported for tumor genomes, little is known on how they compare with alterations present in non-tumor genomes. A comparison of the two would be crucial to better characterize the genetic alterations driving tumorigenesis. We sequenced the genomes of a lymphoblastoid (HCC1954BL) and a breast tumor (HCC1954) cell line derived from the same patient and compared the somatic alterations present in both. The lymphoblastoid genome presents a comparable number and similar spectrum of nucleotide substitutions to that found in the tumor genome. However, a significant difference in the ratio of non-synonymous to synonymous substitutions was observed between both genomes (P = 0.031). Protein–protein interaction analysis revealed that mutations in the tumor genome preferentially affect hub-genes (P = 0.0017) and are co-selected to present synergistic functions (P < 0.0001). KEGG analysis showed that in the tumor genome most mutated genes were organized into signaling pathways related to tumorigenesis. No such organization or synergy was observed in the lymphoblastoid genome. Our results indicate that endogenous mutagens and replication errors can generate the overall number of mutations required to drive tumorigenesis and that it is the combination rather than the frequency of mutations that is crucial to complete tumorigenic transformation

Distinct patterns of somatic alterations in a lymphoblastoid and a tumor genome derived from the same individual

Although patterns of somatic alterations have been reported for tumor genomes, little is known on how they compare with alterations present in non-tumor genomes. A comparison of the two would be crucial to better characterize the genetic alterations driving tumorigenesis. We sequenced the genomes of a lymphoblastoid (HCC1954BL) and a breast tumor (HCC1954) cell line derived from the same patient and compared the somatic alterations present in both. The lymphoblastoid genome presents a comparable number and similar spectrum of nucleotide substitutions to that found in the tumor genome. However, a significant difference in the ratio of non-synonymous to synonymous substitutions was observed between both genomes (P = 0.031). Protein–protein interaction analysis revealed that mutations in the tumor genome preferentially affect hub-genes (P = 0.0017) and are co-selected to present synergistic functions (P < 0.0001). KEGG analysis showed that in the tumor genome most mutated genes were organized into signaling pathways related to tumorigenesis. No such organization or synergy was observed in the lymphoblastoid genome. Our results indicate that endogenous mutagens and replication errors can generate the overall number of mutations required to drive tumorigenesis and that it is the combination rather than the frequency of mutations that is crucial to complete tumorigenic transformation

Alternative polyadenylation allows differential negative feedback of human miRNA miR-579 on its host gene ZFR.

About half of the known miRNA genes are located within protein-coding host genes, and are thus subject to co-transcription. Accumulating data indicate that this coupling may be an intrinsic mechanism to directly regulate the host gene's expression, constituting a negative feedback loop. Inevitably, the cell requires a yet largely unknown repertoire of methods to regulate this control mechanism. We propose APA as one possible mechanism by which negative feedback of intronic miRNA on their host genes might be regulated. Using in-silico analyses, we found that host genes that contain seed matching sites for their intronic miRNAs yield longer 32UTRs with more polyadenylation sites. Additionally, the distribution of polyadenylation signals differed significantly between these host genes and host genes of miRNAs that do not contain potential miRNA binding sites. We then transferred these in-silico results to a biological example and investigated the relationship between ZFR and its intronic miRNA miR-579 in a U87 cell line model. We found that ZFR is targeted by its intronic miRNA miR-579 and that alternative polyadenylation allows differential targeting. We additionally used bioinformatics analyses and RNA-Seq to evaluate a potential cross-talk between intronic miRNAs and alternative polyadenylation. CPSF2, a gene previously associated with alternative polyadenylation signal recognition, might be linked to intronic miRNA negative feedback by altering polyadenylation signal utilization

Gene Copy-Number Polymorphism Caused by Retrotransposition in Humans

The era of whole-genome sequencing has revealed that gene copy-number changes caused by duplication and deletion events have important evolutionary, functional, and phenotypic consequences. Recent studies have therefore focused on revealing the extent of variation in copy-number within natural populations of humans and other species. These studies have found a large number of copy-number variants (CNVs) in humans, many of which have been shown to have clinical or evolutionary importance. For the most part, these studies have failed to detect an important class of gene copy-number polymorphism: gene duplications caused by retrotransposition, which result in a new intron-less copy of the parental gene being inserted into a random location in the genome. Here we describe a computational approach leveraging nextgeneration sequence data to detect gene copy-number variants caused by retrotransposition (retroCNVs), and we report the first genome-wide analysis of these variants in humans. We find that retroCNVs account for a substantial fraction of gene copy-number differences between any two individuals. Moreover, we show that these variants may often result in expressed chimeric transcripts, underscoring their potential for the evolution of novel gene functions. By locating the insertion sites of these duplicates, we are able to show that retroCNVs have had an important role in recent human adaptation, and we also uncover evidence that positive selection may currently be driving multiple retroCNVs toward fixation. Together these findings imply that retroCNVs are an especially important class of polymorphism, and that futur

Bioinformatics and biomolecular analyses indicate a role for APA in regulation of negative feedback.

<p>A) Comparison of APA-sites for HT miRNA host genes and NT miRNA host genes. B) After CPSF2 silencing HT miRNA host gene UTRs display a different poly(A)-site usage pattern compared to NT miRNA host gene UTRs and regular protein-coding genes’ UTRs. C) The motif discovered in upregulated APA regions after CPSF2 silencing resembles the two canonical polyadenylation sites. D) Distribution of canonical poly(A) signals across the 32UTR of HT miRNA host genes and E) NT miRNA host genes.</p

miR-579 targets its host, ZFR, and the APA associated gene CPSF2.

<p>A) Schematic diagram of the <i>ZFR</i> gene. B) Schematic diagram of the ZFR 32UTR including polyadenylation sites and the seed matching site for miR-579. C) U87 cells were co-transfected with reporter constructs containing wildtype ZFR-32UTR or ZFR-32UTR lacking the miR-579 binding site (mut 32UTR) along with pre-miR-579 or negative control (NC). Results are expressed as Rluc/Fluc ratio relative to NC (mean ± 95% CI; n = 6; *, p < 0.05). D) In U87 cells transiently transfected with scrambled control or pre-miR-579, ZFR and CPSF2 mRNA expression was analyzed by quantitative RT-PCR. Values are mean ± 95% CI; n = 5; *, p < 0.05. E) Western blot analysis of the same samples using specific antibodies as indicated (β-Actin served as loading control; one representative experiment of three is shown). F) In U87 cells, expression changes of the long (miRNA binding site containing; red) and short (without miRNA binding site; blue) alternatively polyadenylated UTRs after transfection with pre-miR-579 or with scrambled control was determined by quantitative RT-PCR. Values are shown as miR-579 transfection relative to scrambled control (n = 5; *, p < 0.05).</p

Model of intronic negative feedback regulation.

<p>After coexpression of miRNA and host gene, the miRNA directly regulates its host gene as well as CPSF2. After removal of CPSF2 the polyadenylation-complex is biased towards recognition of canonical sites. In the next transcription cycle, the canonical site that precedes the miRNA binding site is utilized. Hence, regulation of the host gene by its intronic miRNA is disabled.</p

HT miRNA host genes with significant 3´UTR changes after CPSF2-silencing.

<p>HT miRNA host genes with significant 3´UTR changes after CPSF2-silencing.</p