S, implying that endoreduplication occurred 32?60 of the way through the mutational history of this genome. Interpretation of mutation timing depends on the accuracy of our earlier and later classification of mutations. We were confident that the tumor had undergone endoreduplication as it showed two characteristic signatures of this phenomenon: multiple duplicated rearrangements and multiple duplicated homozygous regions (Fig. 2). Given that there had been an endoreduplication, we reconstructed the main steps of HCC1187 karyotype evolution by assuming that the simplest possible sequence of events had happened. Implicit was the assumption that, as far as possible, all duplications had occurred at endoreduplication. The INCB039110 web deduced sequence of chromosome changes (Fig. 3) was consistent with monosomic evolution (Fig. 1). Three duplications could not be explained by endoreduplication: these were three chromosomeNon-random Timing of Mutation SubsetsThe distribution of mutations between earlier and later could uncover selective pressure for a mutation to occur at a particular stage in tumor development, or a change in the level of genetic instability. We therefore estimated the number of random and non-randomly timed mutations given the proportions of different mutation classes above.Timing of Mutations in a Breast Cancer GenomeTiming of Mutations in a Breast Cancer GenomeFigure 4. Point mutations on chromosome 6, and whether they occurred before or after endoreduplication. A) Deducing the parental origin of chromosome 6 segments: the simplest explanation for the allele combinations (blue and red lines on the aCGH plot) in terms of parental origin. Both copies of chromosome 6 I (chromosome 6 fragments are designated 6 I, 6A, 6D as in ref. [12]) originate from parent 1 and the chromosome 6 segments of 6A and 6D originate from parent 2. Several small copy number steps are omitted for clarity. B) Sequence traces show whether mutations are on each isolated chromosome. HSD17B8: Chromosome 6I (2 copies) homozygous G.T mutation (black arrow); chromosome 6A and 6D, no mutation. NCB5OR: Chromosome 6, heterozygous mutant (black arrow). C) The likely evolution of the segments of chromosome 6: unbalanced translocation of one copy of chromosome 6 was followed by duplication of both chromosomes 23148522 during endoreduplication. HSD17B8 was mutated on each copy of chromosome 6I, but not on 6A or 6D, while NCB5OR/CYB5R4 was mutated on only one copy of chromosome 6I. The preendoreduplication state was likely to be one normal copy of chromosome 6 with the other having a mutation in HSD17B8 and having suffered unbalanced translocation. The NCB5OR/CYB5R4 mutation occurred after endoreduplication. doi:10.1371/journal.pone.0064991.gsegments of the same parental origin that were present in three copies. The simplest route to these triplications was via endoreduplication followed by an additional single-chromosome duplication. A few steps in the evolution may have been more complex, but this would not have altered the earlier versus later classification very often. Specifically, if all three triplicated chromosomes had taken a more complex evolutionary route (perhaps duplication followed by endoreduplication, followed by loss), the classification of no more than three point mutations could be affected, moving them from the later category to the `undetermined’ class. Some mutations were omitted from Title Loaded From File analysis. These were from the complex regions of 10 p and 11 q where the paren.S, implying that endoreduplication occurred 32?60 of the way through the mutational history of this genome. Interpretation of mutation timing depends on the accuracy of our earlier and later classification of mutations. We were confident that the tumor had undergone endoreduplication as it showed two characteristic signatures of this phenomenon: multiple duplicated rearrangements and multiple duplicated homozygous regions (Fig. 2). Given that there had been an endoreduplication, we reconstructed the main steps of HCC1187 karyotype evolution by assuming that the simplest possible sequence of events had happened. Implicit was the assumption that, as far as possible, all duplications had occurred at endoreduplication. The deduced sequence of chromosome changes (Fig. 3) was consistent with monosomic evolution (Fig. 1). Three duplications could not be explained by endoreduplication: these were three chromosomeNon-random Timing of Mutation SubsetsThe distribution of mutations between earlier and later could uncover selective pressure for a mutation to occur at a particular stage in tumor development, or a change in the level of genetic instability. We therefore estimated the number of random and non-randomly timed mutations given the proportions of different mutation classes above.Timing of Mutations in a Breast Cancer GenomeTiming of Mutations in a Breast Cancer GenomeFigure 4. Point mutations on chromosome 6, and whether they occurred before or after endoreduplication. A) Deducing the parental origin of chromosome 6 segments: the simplest explanation for the allele combinations (blue and red lines on the aCGH plot) in terms of parental origin. Both copies of chromosome 6 I (chromosome 6 fragments are designated 6 I, 6A, 6D as in ref. [12]) originate from parent 1 and the chromosome 6 segments of 6A and 6D originate from parent 2. Several small copy number steps are omitted for clarity. B) Sequence traces show whether mutations are on each isolated chromosome. HSD17B8: Chromosome 6I (2 copies) homozygous G.T mutation (black arrow); chromosome 6A and 6D, no mutation. NCB5OR: Chromosome 6, heterozygous mutant (black arrow). C) The likely evolution of the segments of chromosome 6: unbalanced translocation of one copy of chromosome 6 was followed by duplication of both chromosomes 23148522 during endoreduplication. HSD17B8 was mutated on each copy of chromosome 6I, but not on 6A or 6D, while NCB5OR/CYB5R4 was mutated on only one copy of chromosome 6I. The preendoreduplication state was likely to be one normal copy of chromosome 6 with the other having a mutation in HSD17B8 and having suffered unbalanced translocation. The NCB5OR/CYB5R4 mutation occurred after endoreduplication. doi:10.1371/journal.pone.0064991.gsegments of the same parental origin that were present in three copies. The simplest route to these triplications was via endoreduplication followed by an additional single-chromosome duplication. A few steps in the evolution may have been more complex, but this would not have altered the earlier versus later classification very often. Specifically, if all three triplicated chromosomes had taken a more complex evolutionary route (perhaps duplication followed by endoreduplication, followed by loss), the classification of no more than three point mutations could be affected, moving them from the later category to the `undetermined’ class. Some mutations were omitted from analysis. These were from the complex regions of 10 p and 11 q where the paren.
