BACKGROUND
the progression from normal cell, subject to stringent growth controls, to an unregulated tumor cell is a stepwise process of accumulating mutations and rearrangements in the genome  <cit> , which may disrupt, inhibit or deregulate genes, or create novel fusion genes.
however, to distinguish genetic alterations which confer strong advantages to the tumor cell  from the more numerous neutral aberrations  is a difficult task.
next-generation sequencing has revealed somatic mutations that may contribute to breast tumorigenesis  <cit> .
several whole genome sequencing studies aiming at detection of chromosomal alterations in cancer genomes have been carried out in the past few years thanks to the advances in massively parallel sequencing technology  <cit> .
the widely adopted method for clinical classification of breast cancer subtypes is usually based on immunohistochemical  analysis of estrogen receptor , progesterone receptor  and human epidermal receptor  <dig> , categorizing breast carcinomas into hormone receptor positive and her <dig> negative , hormone receptor positive and her2+ , hormone receptor negative and her <dig> positive  and triple negative breast cancer  subtypes.
ihc classification is of great value in clinical practice to predict disease outcome as well as assign suitable targeted treatments to patients.
hormone receptor negative breast cancers, composed by hr-/her2+ and tnbc subtypes, often correlate with poor prognosis  <cit> .
her2+ cases often respond well to treatment with trastuzumab, which is an inhibitor of her2-dependent signaling.
however, there is no efficient targeted therapy for tnbcs  <cit> .
there are at least five molecular subtypes of breast cancers defined by gene expression profiles: luminal a, luminal b, her2-enriched, basal-like and normal-like.
hr + tumors are frequently of luminal a or luminal b subtypes, while receptor negative tumors are most frequently of her2-enriched or basal-like subtypes  <cit> .
recent large-scale breast cancer genome studies have revealed that each molecular subtype has specific pattern of genomic alterations and notably, tumors of her2-enriched and basal-like subtypes harbor many more rearrangements than the luminal a subtype, which primarily consists of receptor positive breast tumors  <cit> .
in this work, we performed whole-genome sequencing on  <dig> hormone receptor negative breast cancers  to detect somatic gene rearrangements.
long-insert mate pair sequencing with ~ <dig>  kb insert size was chosen for increased detectability.
pcr and sanger sequencing confirmation of selected structural variants identified  <dig> novel somatic gene rearrangements and  <dig> genes directly affected by these alterations.
we also demonstrate the potential biological functions of some affected genes by these rearrangements by rna interference  in breast cell lines.
RESULTS
landscapes of rearrangement
thirteen breast cancers were sequenced with life technologies solid  <dig>  from which a total of 119 mb mate pair reads were obtained, corresponding to an average nucleotide coverage of ~ <dig> -fold and an average clone coverage of 8-fold per sample.
two additional breast tumors were sequenced with solid  <dig> to a read depth of 3-fold nucleotide coverage and 80-fold clone coverage on average.
structural variations  in the form of deletions, insertion, chromosomal translocations or inversions were observed in 8% of all mate pairs .
the proportions and types of svs vary among tumors, with two tumors having thousands of insertions  while the other samples have much fewer, ranging from  <dig> to  <dig>  in total,  <dig> putative rearrangements were selected for validation , and  <dig>  yielded products consistent with the predictions from the mate pair sequencing.
of these,  <dig> were also found in patient-matched normal tissue suggesting the presence of constitutional svs, while  <dig> were observed only in tumor tissue and considered to be true somatic rearrangements .
somatic svs in individual tumors, including  <dig> deletions,  <dig> inversions and  <dig> interchromosomal translocations, are shown in additional file  <dig>  interestingly, in one tumor sample , we observed at least  <dig> validated translocations between chromosome  <dig> and  <dig>  which may imply chromothripsis  <cit> .
due to the size of mate-pair inserts, insertions could not be validated by pcr.
attempted denotes the number of rearrangements attempted to validate, and constitutional and somatic denote the number of validated rearrangements in the normal and tumor samples validated by pcr followed by sanger sequencing.
chr  <dig>  the lower numbered chromosome in a rearrangement; breakpoint  <dig>  the breakpoint position on chr  <dig> determined by sanger sequencing; chr  <dig>  the other chromosome in a rearrangement; breakpoint  <dig>  the breakpoint position on chr  <dig>  chromosome coordinates are based on human genome build hg <dig>
genes affected by rearrangements
twenty-nine genes were predicted to be directly affected by the  <dig> validated somatic rearrangements, including genes previously reported to be altered in cancer as well as genes that have not yet been related to cancer .
using gene ontology   <cit>  as a reference for potential gene functions , we discovered that these  <dig> affected genes are involved in multiple biological processes including epigenetic regulation , cell mitosis , signal transduction  and others.
in order to gain insight into the functional role of some of these genes in tumor cell growth, we performed small interfering rna  knock-down analysis targeting candidate genes cltc , epha <dig> , ska <dig> , ddx <dig>  box polypeptide 10) and tnik .
we transfected sirna targeting each gene into human breast adenocarcinoma cell line mcf- <dig> and the mammary epithelial cell line mcf-10a.
downregulation of cltc, ska <dig> and ddx <dig> expression was confirmed by rt-pcr in both cell lines .
however, we failed to evaluate the effectiveness of knock-down for genes epha <dig> and tnik due to poor quality of primers.
relative cell growth  was  <dig>  ±  <dig> ,  <dig>  ±  <dig> ,  <dig>  ±  <dig> ,  <dig>  ±  <dig>  and  <dig>  ±  <dig>  in cltc, epha <dig>  ska <dig>  ddx <dig> and tnik knock-down mcf- <dig> cell lines, respectively.
relative cell growth in transfected mcf-10a cell lines was  <dig>  ±  <dig> ,  <dig>  ±  <dig> ,  <dig>  ±  <dig> ,  <dig>  ±  <dig>  and  <dig>  ±  <dig>  for cltc, epha <dig>  ska <dig>  ddx <dig> and tnik respectively.
suppression of any of these genes led to growth inhibition in both cell lines tested .
consistent with previous studies  <cit> , we did not observe frequently recurrent rearrangements.
the only events that disrupted the same genes in two tumors were translocations t, which breakpoints were located within the genes ska <dig> and ddx <dig>  ska <dig> is required for spindle checkpoint silencing, the maintenance of chromosome cohesion in mitosis and metaphase to anaphase progression  <cit> , whereas ddx <dig> encodes a dead-box rna helicase and is known to form an nup98-ddx <dig> fusion oncogene in leukemia  <cit> .
in addition to decreased cell growth, we observed a higher percentage of cells with apoptotic nuclear morphology after suppression of ddx <dig> expression in these mcf- <dig>  and mcf-10a  cell line.
in cells treated with sirna targeting ska <dig>  apoptotic nuclei were observed at a similar or slightly greater frequency compared to control .
we also investigated the formation of micronuclei.
consistent with previous observations  <cit> , about 5% of mcf- <dig> cells and 2-3% mcf-10a cells harbored micronuclei, while suppression of either ddx <dig> or ska <dig> expression did not lead to any significant change in this frequency .
these findings indicate that the ska3/ddx <dig> alterations may have potential roles in tumor development, and ddx <dig> may be involved in pathways mediating cell apoptosis.
we also observed and validated a putative in-frame gene fusion of plekha <dig> and asic <dig> as a result of a translocation between chromosomes  <dig> and  <dig>  this rearrangement was constitutional rather than somatic.
nevertheless, we cannot exclude the intriguing possibility that this fusion could be driving tumorigenesis, since somatic point mutations and rearrangements in asic <dig> have been observed in previous studies  <cit> .
discussion
long-insert mate pair sequencing for detecting gene rearrangements
in this study we chose to perform long-insert  mate pair sequencing to comprehensively identify structural alterations in receptor-negative breast cancers.
potential advantages of the approach include higher sensitivity and higher likelihood of detecting svs within repetitive regions.
long insert lengths also reduce the need for high sequence coverage, especially when searching for potential breakpoints in the chromosomes as consequences of svs.
however, one possible drawback of long inserts could arguably be an increased difficulty of validation, since pcr product sizes may be prohibitively large when few reads span a breakpoint.
in this study, the majority  of validated rearrangements were interchromosomal, which underly the fact that most  true deletions were confirmed to be germline variants instead of somatic events, whereas a smaller number of translocations  were present in matched normal tissues.
however, this proportion differs from a previous investigation where interchromosomal events only composed less than 10% of all kinds of structural variations.
despite the limited sample size in both studies and different classification system of rearrangements, the ratios of interchromosomal events to intrachromosomal deletions and inversions in these two studies vary to a large extent:  <dig>  in our study and  <dig>  in  <cit> .
this discrepancy may be explained by the selection of rearrangements for validation in this study, since we only attempted to confirm the svs that occurred in or within two insert lengths of refseq genes and within two insert lengths of similar svs in other tumors.
it might also indicate the difference in the detectability of distinct types of rearrangement between these two studies due to approaches used .
potential function in cell growth and survival revealed by sirna knock-down analysis
results of sirna experiments in cell lines indicate that the genes cltc, epha <dig>  ska <dig>  ddx <dig> and tnik might be functional in cell growth, and ddx <dig> is probably involved in cell apoptosis.
however, the cell lines mcf- <dig> and mcf-10a used in this analysis may be suboptimal since they do not represent receptor negative breast cancers.
therefore, additional cell lines, especially receptor negative breast cancer cell lines will need to be studied to ultimately determine the function of these genes in breast cancer development.
recurrently affected genes in other cancer genome sequencing studies
to identify potential recurrent somatic rearrangements in breast cancer, we compared validated somatic svs in this study with findings from several recently published breast cancer genome reports  <cit> .
none of the validated svs were observed in any other previous studies, demonstrating that recurrent somatic rearrangements are very rare in breast cancers.
however, some genes were affected by somatic svs in more than one breast cancer.
somatic deletions in eya <dig> were previously reported in four er + breast tumors and one tnbc  <cit> , revealing a combined prevalence of 7% .
eya <dig> encodes a transcription factor where mutations have been associated with the branchio-oto-renal syndrome and other developmental abnormalities  <cit> .
dscam was affected by complex svs including amplification, inversion, deletion and interchromosomal translocation in three additional breast tumors  <cit> .
dscam  activates jnk and p <dig> map kinases and is important for axon guidance in the central neuron system  <cit> .
amplification and interchromosomal translocation of cltc encoding the heavy chain of clathrin, which is required for the function of the mitotic spindle  <cit> , were reported in two breast tumors  <cit> .
cltc also forms fusion genes in 25-30% of lymphomas and myofibroblastic tumors  <cit> .
deletion, amplification and interchromosomal translocation of kiaa <dig> were previously discovered in two breast cancers  <cit> .
moreover, genes epha <dig>  lrba , thsd <dig> , ddx <dig>  gucy1a <dig> , jmjd1c , kdm3b -specific demethylase 3b), ksr <dig> , pfkm , pip4k2a , rora , smarcc <dig> , spred <dig> , tmem <dig>  and tnik have been reported for deletions in at least one breast tumor in previous studies  <cit> , suggesting that the roles of these genes in cancer merit further investigation.
interestingly, jmjd1c was observed downregulated in breast cancers compared to normal tissues, suggesting it as a potential tumor suppressor gene  <cit> .
lrba was found upregulated in several different cancers including er + breast tumor, and lrba knockdown promotes cancer cell apoptosis  <cit> .
comparison of breakpoint location to array painting study and fragile sites
we also compared the translocation breakpoints in the present study to the total non-redundant translocation breakpoint regions  previously revealed by array painting of three breast cancer cell lines hcc <dig>  hcc <dig> and zr-75- <dig>  <cit> .
of our  <dig> breakpoints in  <dig> translocations, we observe 38% within the translocation breakpoint regions, significantly higher than the expected 15% .
out of  <dig> non-redundant translocation breakpoint regions,  <dig> co-occur at least once with putative translocations in this study , altogether suggesting a correlation in breakpoint regions of translocations in breast cancers between our study and the previous screen, which might reveal two possible scenarios; first, common defects in the dna repair mechanisms in breast cancer may led to similar patterns of chromosome breakage, or second, the breakpoints represent driver rearrangements conferring a selective advantage in carcinogenesis.
we also studied the overlap between the translocation breakpoints in this study and the fragile sites of chromosomes  previously reported by debacker and coworkers  <cit> .
we observed 11% of total breakpoints occur within the fragile regions, as compared to the expected  <dig> %, which suggests that there is no significant correlation between double strand breaks and fragile sites .
therefore, the possibility that the correlation between mate pair sequencing and array painting is caused by intrinsic characteristics of chromosomes could be ruled out.
enrichment of consensus cancer genes in genes affected by rearrangements
while the mechanisms and routes to breast cancer may be complex, the accumulation of sequenced tumors will eventually lead to a clearer understanding of the necessary genetic rearrangements.
this work focuses on  <dig> samples, and we identify alterations that recur not only within our samples, but also with previous large scale studies  <cit> .
moreover, among the  <dig> genes disrupted directly by svs,  <dig> genes are included in the human cancer-gene census  <cit> : chn <dig>  cltc, ddx <dig> and mecom, suggesting an enrichment of consensus cancer genes in our results .
however, it should be noted that these genes may not represent all the genes affected by the rearrangements, since only the validated svs were considered here and we only attempted to validate deletions, inversions and translocations that occurred in or near refseq genes.
the distribution of svs varies markedly, reflecting the unique genetic composition of each tumor.
however, the most striking difference is in the number of insertions, where two samples  have thousands of insertions, in contrast to much lower numbers in other samples .
this does not seem to be the consequence of differences in sequence coverage, since the two most deeply sequenced samples harbor only  <dig> insertions in total.
brca <dig> and brca <dig> mutational analyses detect no germline mutations in the two patients with extremely large numbers of insertions, indicating that this feature is not caused by deficiency in dna repair mechanisms as a result of brca <dig> or brca <dig> mutations.
the bulk of the insertions in samples 120 t and 150 t tend to be shorter with an average insertion length of roughly 600 bp, compared to about 1000 bp in other samples , which may suggest a different mutagenesis mechanism in these two samples.
CONCLUSIONS
in this study we identify gene rearrangements in receptor negative breast cancer genomes using long-insert whole-genome mate pair sequencing.
somatic rearrangements disrupting genes composed by both known cancer genes and genes not previously correlated with cancer have been validated.
these genes include epigenetic regulators, genes involved in mitosis and multiple signaling pathways and other genes whose functions are largely unknown.
consistent with previous studies  <cit> , we did not observe frequently recurrent rearrangements, which verify the fact that breast cancer is a highly heterogeneous disease that a large number of low-frequency rearrangements may synergistically contribute to its development.

 the progression from normal cell, subject to stringent growth controls, to an unregulated tumor cell is a stepwise process of accumulating mutations and rearrangements in the genome, which may disrupt, inhibit or deregulate genes, or create novel fusion genes. however, to distinguish genetic alterations which confer strong advantages to the tumor cell from the more numerous neutral aberrations is a difficult task. next-generation sequencing has revealed somatic mutations that may contribute to breast tumorigenesis. several whole genome sequencing studies aiming at detection of chromosomal alterations in cancer genomes have been carried out in the past few years thanks to the advances in massively parallel sequencing technology. the widely adopted method for clinical classification of breast cancer subtypes is usually based on immunohistochemical analysis of estrogen receptor, progesterone receptor and human epidermal receptor, categorizing breast carcinomas into hormone receptor positive and her negative, hormone receptor positive and her2+, hormone receptor negative and her positive and triple negative breast cancer subtypes. ihc classification is of great value in clinical practice to predict disease outcome as well as assign suitable targeted treatments to patients. hormone receptor negative breast cancers, composed by hr-/her2+ and tnbc subtypes, often correlate with poor prognosis. her2+ cases often respond well to treatment with trastuzumab, which is an inhibitor of her2-dependent signaling. however, there is no efficient targeted therapy for tnbcs. there are at least five molecular subtypes of breast cancers defined by gene expression profiles: luminal a, luminal b, her2-enriched, basal-like and normal-like. hr + tumors are frequently of luminal a or luminal b subtypes, while receptor negative tumors are most frequently of her2-enriched or basal-like subtypes. recent large-scale breast cancer genome studies have revealed that each molecular subtype has specific pattern of genomic alterations and notably, tumors of her2-enriched and basal-like subtypes harbor many more rearrangements than the luminal a subtype, which primarily consists of receptor positive breast tumors. in this work, we performed whole-genome sequencing on hormone receptor negative breast cancers to detect somatic gene rearrangements. long-insert mate pair sequencing with ~  kb insert size was chosen for increased detectability. pcr and sanger sequencing confirmation of selected structural variants identified novel somatic gene rearrangements and genes directly affected by these alterations. we also demonstrate the potential biological functions of some affected genes by these rearrangements by rna interference in breast cell lines.  landscapes of rearrangement thirteen breast cancers were sequenced with life technologies solid from which a total of 119 mb mate pair reads were obtained, corresponding to an average nucleotide coverage of ~ -fold and an average clone coverage of 8-fold per sample. two additional breast tumors were sequenced with solid to a read depth of 3-fold nucleotide coverage and 80-fold clone coverage on average. structural variations in the form of deletions, insertion, chromosomal translocations or inversions were observed in 8% of all mate pairs. the proportions and types of svs vary among tumors, with two tumors having thousands of insertions while the other samples have much fewer, ranging from to in total, putative rearrangements were selected for validation, and yielded products consistent with the predictions from the mate pair sequencing. of these, were also found in patient-matched normal tissue suggesting the presence of constitutional svs, while were observed only in tumor tissue and considered to be true somatic rearrangements. somatic svs in individual tumors, including deletions, inversions and interchromosomal translocations, are shown in additional file interestingly, in one tumor sample, we observed at least validated translocations between chromosome and which may imply chromothripsis. due to the size of mate-pair inserts, insertions could not be validated by pcr. attempted denotes the number of rearrangements attempted to validate, and constitutional and somatic denote the number of validated rearrangements in the normal and tumor samples validated by pcr followed by sanger sequencing. chr the lower numbered chromosome in a rearrangement; breakpoint the breakpoint position on chr determined by sanger sequencing; chr the other chromosome in a rearrangement; breakpoint the breakpoint position on chr chromosome coordinates are based on human genome build hg genes affected by rearrangements twenty-nine genes were predicted to be directly affected by the validated somatic rearrangements, including genes previously reported to be altered in cancer as well as genes that have not yet been related to cancer. using gene ontology as a reference for potential gene functions, we discovered that these affected genes are involved in multiple biological processes including epigenetic regulation, cell mitosis, signal transduction and others. in order to gain insight into the functional role of some of these genes in tumor cell growth, we performed small interfering rna knock-down analysis targeting candidate genes cltc, epha, ska, ddx box polypeptide 10) and tnik. we transfected sirna targeting each gene into human breast adenocarcinoma cell line mcf- and the mammary epithelial cell line mcf-10a. downregulation of cltc, ska and ddx expression was confirmed by rt-pcr in both cell lines. however, we failed to evaluate the effectiveness of knock-down for genes epha and tnik due to poor quality of primers. relative cell growth was  ± ,  ± ,  ± ,  ±  and  ±  in cltc, epha ska ddx and tnik knock-down mcf- cell lines, respectively. relative cell growth in transfected mcf-10a cell lines was  ± ,  ± ,  ± ,  ±  and  ±  for cltc, epha ska ddx and tnik respectively. suppression of any of these genes led to growth inhibition in both cell lines tested. consistent with previous studies, we did not observe frequently recurrent rearrangements. the only events that disrupted the same genes in two tumors were translocations t, which breakpoints were located within the genes ska and ddx ska is required for spindle checkpoint silencing, the maintenance of chromosome cohesion in mitosis and metaphase to anaphase progression, whereas ddx encodes a dead-box rna helicase and is known to form an nup98-ddx fusion oncogene in leukemia. in addition to decreased cell growth, we observed a higher percentage of cells with apoptotic nuclear morphology after suppression of ddx expression in these mcf- and mcf-10a cell line. in cells treated with sirna targeting ska apoptotic nuclei were observed at a similar or slightly greater frequency compared to control. we also investigated the formation of micronuclei. consistent with previous observations, about 5% of mcf- cells and 2-3% mcf-10a cells harbored micronuclei, while suppression of either ddx or ska expression did not lead to any significant change in this frequency. these findings indicate that the ska3/ddx alterations may have potential roles in tumor development, and ddx may be involved in pathways mediating cell apoptosis. we also observed and validated a putative in-frame gene fusion of plekha and asic as a result of a translocation between chromosomes and this rearrangement was constitutional rather than somatic. nevertheless, we cannot exclude the intriguing possibility that this fusion could be driving tumorigenesis, since somatic point mutations and rearrangements in asic have been observed in previous studies. discussion long-insert mate pair sequencing for detecting gene rearrangements in this study we chose to perform long-insert mate pair sequencing to comprehensively identify structural alterations in receptor-negative breast cancers. potential advantages of the approach include higher sensitivity and higher likelihood of detecting svs within repetitive regions. long insert lengths also reduce the need for high sequence coverage, especially when searching for potential breakpoints in the chromosomes as consequences of svs. however, one possible drawback of long inserts could arguably be an increased difficulty of validation, since pcr product sizes may be prohibitively large when few reads span a breakpoint. in this study, the majority of validated rearrangements were interchromosomal, which underly the fact that most true deletions were confirmed to be germline variants instead of somatic events, whereas a smaller number of translocations were present in matched normal tissues. however, this proportion differs from a previous investigation where interchromosomal events only composed less than 10% of all kinds of structural variations. despite the limited sample size in both studies and different classification system of rearrangements, the ratios of interchromosomal events to intrachromosomal deletions and inversions in these two studies vary to a large extent: in our study and in. this discrepancy may be explained by the selection of rearrangements for validation in this study, since we only attempted to confirm the svs that occurred in or within two insert lengths of refseq genes and within two insert lengths of similar svs in other tumors. it might also indicate the difference in the detectability of distinct types of rearrangement between these two studies due to approaches used. potential function in cell growth and survival revealed by sirna knock-down analysis results of sirna experiments in cell lines indicate that the genes cltc, epha ska ddx and tnik might be functional in cell growth, and ddx is probably involved in cell apoptosis. however, the cell lines mcf- and mcf-10a used in this analysis may be suboptimal since they do not represent receptor negative breast cancers. therefore, additional cell lines, especially receptor negative breast cancer cell lines will need to be studied to ultimately determine the function of these genes in breast cancer development. recurrently affected genes in other cancer genome sequencing studies to identify potential recurrent somatic rearrangements in breast cancer, we compared validated somatic svs in this study with findings from several recently published breast cancer genome reports. none of the validated svs were observed in any other previous studies, demonstrating that recurrent somatic rearrangements are very rare in breast cancers. however, some genes were affected by somatic svs in more than one breast cancer. somatic deletions in eya were previously reported in four er + breast tumors and one tnbc, revealing a combined prevalence of 7%. eya encodes a transcription factor where mutations have been associated with the branchio-oto-renal syndrome and other developmental abnormalities. dscam was affected by complex svs including amplification, inversion, deletion and interchromosomal translocation in three additional breast tumors. dscam activates jnk and p map kinases and is important for axon guidance in the central neuron system. amplification and interchromosomal translocation of cltc encoding the heavy chain of clathrin, which is required for the function of the mitotic spindle, were reported in two breast tumors. cltc also forms fusion genes in 25-30% of lymphomas and myofibroblastic tumors. deletion, amplification and interchromosomal translocation of kiaa were previously discovered in two breast cancers. moreover, genes epha lrba, thsd, ddx gucy1a, jmjd1c, kdm3b -specific demethylase 3b), ksr, pfkm, pip4k2a, rora, smarcc, spred, tmem and tnik have been reported for deletions in at least one breast tumor in previous studies, suggesting that the roles of these genes in cancer merit further investigation. interestingly, jmjd1c was observed downregulated in breast cancers compared to normal tissues, suggesting it as a potential tumor suppressor gene. lrba was found upregulated in several different cancers including er + breast tumor, and lrba knockdown promotes cancer cell apoptosis. comparison of breakpoint location to array painting study and fragile sites we also compared the translocation breakpoints in the present study to the total non-redundant translocation breakpoint regions previously revealed by array painting of three breast cancer cell lines hcc hcc and zr-75-. of our breakpoints in translocations, we observe 38% within the translocation breakpoint regions, significantly higher than the expected 15%. out of non-redundant translocation breakpoint regions, co-occur at least once with putative translocations in this study, altogether suggesting a correlation in breakpoint regions of translocations in breast cancers between our study and the previous screen, which might reveal two possible scenarios; first, common defects in the dna repair mechanisms in breast cancer may led to similar patterns of chromosome breakage, or second, the breakpoints represent driver rearrangements conferring a selective advantage in carcinogenesis. we also studied the overlap between the translocation breakpoints in this study and the fragile sites of chromosomes previously reported by debacker and coworkers. we observed 11% of total breakpoints occur within the fragile regions, as compared to the expected %, which suggests that there is no significant correlation between double strand breaks and fragile sites. therefore, the possibility that the correlation between mate pair sequencing and array painting is caused by intrinsic characteristics of chromosomes could be ruled out. enrichment of consensus cancer genes in genes affected by rearrangements while the mechanisms and routes to breast cancer may be complex, the accumulation of sequenced tumors will eventually lead to a clearer understanding of the necessary genetic rearrangements. this work focuses on samples, and we identify alterations that recur not only within our samples, but also with previous large scale studies. moreover, among the genes disrupted directly by svs, genes are included in the human cancer-gene census: chn cltc, ddx and mecom, suggesting an enrichment of consensus cancer genes in our results. however, it should be noted that these genes may not represent all the genes affected by the rearrangements, since only the validated svs were considered here and we only attempted to validate deletions, inversions and translocations that occurred in or near refseq genes. the distribution of svs varies markedly, reflecting the unique genetic composition of each tumor. however, the most striking difference is in the number of insertions, where two samples have thousands of insertions, in contrast to much lower numbers in other samples. this does not seem to be the consequence of differences in sequence coverage, since the two most deeply sequenced samples harbor only insertions in total. brca and brca mutational analyses detect no germline mutations in the two patients with extremely large numbers of insertions, indicating that this feature is not caused by deficiency in dna repair mechanisms as a result of brca or brca mutations. the bulk of the insertions in samples 120 t and 150 t tend to be shorter with an average insertion length of roughly 600 bp, compared to about 1000 bp in other samples, which may suggest a different mutagenesis mechanism in these two samples.  in this study we identify gene rearrangements in receptor negative breast cancer genomes using long-insert whole-genome mate pair sequencing. somatic rearrangements disrupting genes composed by both known cancer genes and genes not previously correlated with cancer have been validated. these genes include epigenetic regulators, genes involved in mitosis and multiple signaling pathways and other genes whose functions are largely unknown. consistent with previous studies, we did not observe frequently recurrent rearrangements, which verify the fact that breast cancer is a highly heterogeneous disease that a large number of low-frequency rearrangements may synergistically contribute to its development. 
other rearrangements were found to affect genes in pathways involved in epigenetic regulation, mitosis and signal transduction, underscoring their potential role in breast tumorigenesis. to identify novel somatic chromosomal rearrangements, we determined the genome structures of hormone-receptor negative breast tumors by long-insert mate pair massively parallel sequencing. moreover, downregulation of ddx in breast cancer cells lead to an increased frequency of apoptotic nuclear morphology.
 chromosomal rearrangements in the form of deletions, insertions, inversions and translocations are frequently observed in breast cancer genomes, and a subset of these rearrangements may play a crucial role in tumorigenesis. to identify novel somatic chromosomal rearrangements, we determined the genome structures of hormone-receptor negative breast tumors by long-insert mate pair massively parallel sequencing.  we identified and validated somatic structural alterations, including the recurring fusion between genes ddx and ska and translocations involving the epha gene. other rearrangements were found to affect genes in pathways involved in epigenetic regulation, mitosis and signal transduction, underscoring their potential role in breast tumorigenesis. rna interference-mediated suppression of five candidate genes led to inhibition of breast cancer cell growth. moreover, downregulation of ddx in breast cancer cells lead to an increased frequency of apoptotic nuclear morphology.  using whole genome mate pair sequencing and rna interference assays, we have discovered a number of novel gene rearrangements in breast cancer genomes and identified ddx ska epha cltc and tnik as potential cancer genes with impact on the growth and proliferation of breast cancer cells. 