Pedigrees of the breast cancer cases with germline mutations in <i>CHEK2</i>.

<p>The index individuals initially screened are indicated with arrows. All cancer patients marked in bold, and cancers are indicated by type and age at diagnosis. D followed by number indicates the age of death. #, indicate that diagnosis could not be verified from medical documents. Mut −, indicates individuals tested negative for relevant mutations. Mut +, indicates that individuals hold the relevant mutation. The trees have been altered to preserve anonymity, but the meaning of the report is not affected by these alterations. BC, Breast cancer; BD, Blood disease; BLC, Bladder cancer; CC, Colon cancer; EC, Endometri cancer; GC, Gastric cancer; HD, Heart disease; HL, Hodgkins Lymphoma; L, Lymphoma; LAC, Larynx cancer; LC, Lung cancer; LE, Leukemia; LEC, Liver cancer; OC, Ovarian cancer; OL, Oral Lymphoma M; P, Parkinson; PC, Prostate cancer; SA, Sarcoma; SE, Seminom; SI, Carcinoid in small intestine; TBC, Tuberculosis.</p

Kaplan-Meyer analysis of the relapse-free survival of the patients according to mutations.

<p>WT, wild-type; <i>TP53</i>+<i>CHEK2</i> mut, all found mutations in <i>TP53</i> and <i>CHEK2</i>; <i>TP53</i> L2/L3+<i>CHEK2</i> (Arg95Ter) mut, <i>TP53</i> mutations affection L2/L3 domain and <i>CHEK2</i> mutations affecting kinase function; <i>TP53</i>+<i>CHEK2</i> (Ile364Thr), mutations not affecting L2/L3 domains and <i>CHEK2</i> mutations not affecting kinase function. Deaths due to causes other than breast cancer are treated as censored observations. Each “+” mark represents the time one patient was censored. NS, Non significant.</p

Kinase activity of <i>CHEK2</i> mutant's co-transfected with <i>CHEK2</i> wild-type.

<p>A) Kinase assay input of V5-tagged mutant Chk2 and Xpr-tagged wild-type Chk2, monitored by anti-V5 and anti-Xpr based Western blot analysis. B) Autoradiogram showing <i>in vitro</i> kinase activity (Chk2 autophosphorylation and Cdc25 phosphorylation) of Chk2 mutants with co-precipitated Chk2 wild-type.</p

Contribution of co-precipitated Chk2 wild-type to the activity in the <i>in vitro</i> assays.

<p>Transfection of the Arg117Gly mutant with and without Chk2 wild-type, along with Arg95Ter +/− wild-type. Arg117Gly, when transfected alone, does not display higher kinase activity (Cdc25 phosphorylation) than Arg95Ter or negative control. This strongly indicates that the contribution of endogenous Chk2 is non-significant.</p

Clinical response in relation to different parameters

<p><i>P¹</i> with regard to clinical response comparing CR+PR+SD versus PD</p><p><i>P²</i> with regard to clinical response comparing CR+PR versus PD</p>*<p>One of the PD patients has got a mutation both in <i>CHEK2</i> and <i>TP53</i> (L2 domain), this has been taken into consideration under calculation of statistical significance</p>1<p>P, with regard to clinical response comparing CR+PR versus PD; ²P, with regard to clinical response comparing CR+PR+SD versus PD; ^*, One of the PD patients has got a mutation both in <i>CHEK2</i> and <i>TP53</i> (L2 domain), this has been taken into consideration under calculation of statistical significance.</p

Characteristics of <i>CHEK2</i> mutants found and clinical data

<p>¹, The bolded bases indicate the base change; T N M, TNM-classification, AJCC 2002 = UICC 2002, T, size or direct of the primary tumor; N, spread to regional lymph nodes; M, distant metastasis; ³, “F” followed by a number indicates that the patient was free of disease at that number of months of follow-up. “R” followed by a number indicates that the patient was alive at that number of months of follow-up but had suffered a relapse; ˆ, “A” followed by a number indicates that the patient was alive at that number of months of follow-up. “D” followed by a number indicates that the patient died at that number of months of follow-up; AI, Allelic imbalance; NI, Not informative; NA, Not available. “^*” This patient subsequently relapsed with distant metastases at 64 months.</p

Kinase activity of <i>CHEK2</i> mutants.

<p>A) Level of Chk2 mutants immunoprecipitated from U-2-OS cells, used as input for kinase activity assay, monitored by anti-V5 based Western blot analysis. B) Autoradiogram showing <i>in vitro</i> kinase activity of Chk2 mutants with respect to both Chk2 autophosphorylation and Cdc25 phosphorylation. C) Kinase activity of <i>CHEK2</i> mutants normalized for kinase-input, based on band intensities in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003062#pone-0003062-g003" target="_blank">Figures 3A and B</a>.</p

PCR primers for amplification and sequencing of cDNA

<p>PCR primers for amplification and sequencing of cDNA</p

Pulldown-assay for <i>CHEK2</i> mutants.

<p>V5-tagged Chk2 mutants were co-expressed with Xpr-tagged wt-Chk2 in U-2-OS-cells and immunoprecipitation was performed using anti-V5 antibody. Expression of the Chk2 mutants was monitored by anti-V5 based Western blot analysis prior to immunoprecipitation (upper panel). The Chk2 mutant's ability to dimerize with the wild-type protein was detected by anti-Xpr Western blot analysis of the precipitate (lower panel).</p

Distribution of SNP344 genotypes in cancer patients and healthy controls, restricted to individuals carrying the SNP309T-allele.

<p>Distribution of SNP344 genotypes in cancer patients and healthy controls, restricted to individuals carrying the SNP309T-allele.</p