Indication of dysfunction of <i>TP53</i> and β-estradiol centered network in the studied breast cancer cases.

<p>IPA was used to identify the connection between the genes disrupted in all cases (both familial and the cohort consisting of young breast cancer patients). The analysis identified two networks with (A) <i>TP53</i>, β-estradiol and <i>CTNNB1</i> (in green) occupying the central positions, and (B) β-estradiol (in green) occupying the central position. Genes disrupted in breast cancer cases are coloured with red. Solid lines indicate direct molecular interaction and dashed lines indicate indirect molecular interaction.</p

Proportion of rare CNVs in breast cancer cases and controls.

<p>BC = breast cancer.</p>a<p>Observed only in cancer cases, or only in controls.</p>b<p>The genomic loci has annotated genes.</p>c<p>Gene disruptions include rare CNVs having breakpoints within the genes or promoter regions, and rare CNVs which delete the involved genes entirely.</p

Molecular and cellular functions, and diseases and disorders overrepresented among the genes disrupted in familial breast cancer cases.

<p>No particular functions were overrepresented among controls.</p>a<p>Statistically significant false discovery rate (FDR) adjusted <i>P</i>-values; correction for multiple testing was done using the Benjamini-Hochberg method.</p

Targeted Next-Generation Sequencing Identifies a Recurrent Mutation in <i>MCPH1</i> Associating with Hereditary Breast Cancer Susceptibility

<div><p>Breast cancer is strongly influenced by hereditary risk factors, a majority of which still remain unknown. Here, we performed a targeted next-generation sequencing of 796 genes implicated in DNA repair in 189 Finnish breast cancer cases with indication of hereditary disease susceptibility and focused the analysis on protein truncating mutations. A recurrent heterozygous mutation (c.904_916del, p.Arg304ValfsTer3) was identified in early DNA damage response gene, <i>MCPH1</i>, significantly associating with breast cancer susceptibility both in familial (5/145, 3.4%, <i>P</i> = 0.003, OR 8.3) and unselected cases (16/1150, 1.4%, <i>P</i> = 0.016, OR 3.3). A total of 21 mutation positive families were identified, of which one-third exhibited also brain tumors and/or sarcomas (<i>P</i> = 0.0007). Mutation carriers exhibited significant increase in genomic instability assessed by cytogenetic analysis for spontaneous chromosomal rearrangements in peripheral blood lymphocytes (<i>P</i> = 0.0007), suggesting an effect for MCPH1 haploinsufficiency on cancer susceptibility. Furthermore, 40% of the mutation carrier tumors exhibited loss of the wild-type allele. These findings collectively provide strong evidence for <i>MCHP1</i> being a novel breast cancer susceptibility gene, which warrants further investigations in other populations.</p></div

Examples of <i>MCPH1</i> mutation positive families (A-C).

<p>Patients with breast cancer are marked with black half circles. Other cancer types are marked with grey squares. The age at diagnosis, when known, is marked below the cancer type. Individuals genotyped for <i>MCPH1</i> c.904_916del are marked with either a plus (mutation positive) or a minus sign (mutation negative). A slashed pedigree symbol indicates a deceased individual. Triangle indicates the initially studied index patient (BR-0653, BR-0887 and BR-0154, respectively). Abbreviations: Bas: basalioma, Bt: brain tumor, Br: breast cancer, Col: colon cancer, Csu: cancer site unknown, Lar: laryngeal cancer, Ov: ovarian tumor, Pro: prostate cancer, Ut: uterine cancer, Vul: vulvar cancer.</p

Effect of <i>MCPH1</i> c.904_916del mutation at the mRNA and protein level.

<p>(<b>A</b>) Schematic presentation of the MCPH1 protein and the position of the observed truncating mutation. (<b>B</b>) Sequence chromatogram comparisons of genomic DNA and cDNA of heterozygous mutation carriers and a wild-type control. (<b>C</b>) Immunoblotting of 3 mutation carriers and 3 non-carriers with an antibody directed towards the amino-terminus of MCPH1. The representative image of altogether three independent experiments is shown.</p

Occurrence of chromosomal aberrations in blood lymphocyte metaphases of heterozygous <i>MCPH1</i> c.904_916del mutation carriers and healthy controls.

<p>Occurrence of chromosomal aberrations in blood lymphocyte metaphases of heterozygous <i>MCPH1</i> c.904_916del mutation carriers and healthy controls.</p

Family history of cancers of <i>MCPH1</i> c.904_916del positive index cases^a.

<p>Family history of cancers of <i>MCPH1</i> c.904_916del positive index cases<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005816#t002fn004" target="_blank">^a</a>.</p

Overview of the experimental design used in the co-culture assay.

<p>In each experiment, LCLs of the same genotype were seeded into 6-well plates (cells exposed to direct irradiation) and insert plates (bystander cells). Cells in 6-well plates (indicated by white circles) were irradiated with X-ray at doses of 0, 0.01, 0.1, 1, or 2 Gy. Immediately after irradiation, inserts with a permeable filter and containing bystander cells (filled black circles) were placed on top of the cells in the 6-well plates. After 1 or 20 hours co-culture, the directly irradiated and bystander cells were separated and incubated for an additional 24 hours before CA assay and 43 hours before cell viability assay were performed. Total post-irradiation time was 44 or 63 hours in the cell viability assay and 25 or 44 hours in the CA assay.</p

Cell viability in bystander cells.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093211#pone-0093211-g004" target="_blank">Figure 4</a> legend for details.</p