Penetrance analysis of the PALB2 c.1592delT founder mutation.

PURPOSE: PALB2 is a recently identified breast cancer susceptibility gene. We have previously identified in the Finnish population a PALB2 c.1592delT founder truncation mutation that is associated with an increased risk of breast cancer. In the present study, we wanted to assess in more detail the increased risk (hazard ratio, HR) and the age-specific cumulative risk (penetrance) of c.1592delT with regard to susceptibility to breast and other forms of cancer. EXPERIMENTAL DESIGN: Modified segregation analyses fitted under maximum likelihood theory were used to estimate age-specific cumulative risks and HRs using the families of mutation carriers identified from a consecutive series of breast cancer cases unselected for age at onset or family history. RESULTS: We found a substantially increased risk of breast cancer [HR, 6.1; 95% confidence interval (95% CI), 2.2-17.2; P = 0.01] equivalent to a 40% (95% CI, 17-77) breast cancer risk by age 70 years, comparable to that for carriers of mutations in BRCA2. We found marginal evidence (P = 0.06) that the HR for breast cancer decreased with age by 4.2% per year (95% CI, 0.2-8.1), from 7.5-fold at age 30 years to 2.0-fold at age 60 years. CONCLUSIONS: Our results suggest that it may be appropriate to offer PALB2 c.1592delT mutation testing to Finnish women with breast cancer, especially those with an early age at onset or a family history of breast or related cancers, and to offer carriers the option of participation in extended disease surveillance programs.

NAD(P)H:quinone oxidoreductase 1 NQO1*2 genotype (P187S) is a strong prognostic and predictive factor in breast cancer.

NQO1 guards against oxidative stress and carcinogenesis and stabilizes p53. We find that a homozygous common missense variant (NQO1(*)2, rs1800566(T), NM_000903.2:c.558C>T) that disables NQO1 strongly predicts poor survival among two independent series of women with breast cancer (P = 0.002, N = 1,005; P = 0.005, N = 1,162), an effect particularly evident after anthracycline-based adjuvant chemotherapy with epirubicin (P = 7.52 x 10(-6)) and in p53-aberrant tumors (P = 6.15 x 10(-5)). Survival after metastasis was reduced among NQO1(*)2 homozygotes, further implicating NQO1 deficiency in cancer progression and treatment resistance. Consistently, response to epirubicin was impaired in NQO1(*)2-homozygous breast carcinoma cells in vitro, reflecting both p53-linked and p53-independent roles of NQO1. We propose a model of defective anthracycline response in NQO1-deficient breast tumors, along with increased genomic instability promoted by elevated reactive oxygen species (ROS), and suggest that the NQO1 genotype is a prognostic and predictive marker for breast cancer.

ARLTS1 germline variants and the risk for breast, prostate, and colorectal cancer.

Recently, a nonsense alteration Trp149Stop in the ARLTS1 gene was found more frequently in familial cancer cases versus sporadic cancer patients and healthy controls. Here, the role of Trp149Stop or any other ARLTS1 germline variant was evaluated on breast, prostate, and colorectal cancer risk. The whole gene was screened for germline alterations in 855 familial cancer patients. The five observed variants were further screened in 1169 non-familial cancer patients as well as in 809 healthy population controls. The Trp149Stop was found at low frequencies (0.5-1.2%) in all patient subgroups versus 1.6% in controls, and the mutant allele did not co-segregate with disease status in families with multiple affected individuals. The CC genotype in the Cys148Arg variant was slightly more common among both familial and sporadic breast (odds ratio (OR), 1.48; 95% confidence interval (CI), 1.16-1.87; P=0.001) and prostate cancer patients (OR, 1.50; 95% CI, 1.13-1.99; P=0.005) when compared to controls. A novel ARLTS1 variant Gly65Val was found at higher frequency among familial prostate cancer patients (8 of 164, 4.9%) than in controls (13 of 809, 1.6%; OR, 3.14; 95% CI, 1.28-7.70, P=0.016). However, after adjusting for multiple testing, none of these results were still significant. No association was found with any of the variants and colorectal cancer risk. Our results suggest that Trp149Stop is not a predisposition allele in breast, prostate, or colorectal cancer in the Finnish population, and, while the Gly65Val variant may increase familial prostate cancer risk and the Cys148Arg change may affect both breast and prostate cancer risk, the evidence is not strong in these data.

A recurrent mutation in PALB2 in Finnish cancer families.

BRCA1, BRCA2 and other known susceptibility genes account for less than half of the detectable hereditary predisposition to breast cancer. Other relevant genes therefore remain to be discovered. Recently a new BRCA2-binding protein, PALB2, was identified. The BRCA2-PALB2 interaction is crucial for certain key BRCA2 DNA damage response functions as well as its tumour suppression activity. Here we show, by screening for PALB2 mutations in Finland that a frameshift mutation, c.1592delT, is present at significantly elevated frequency in familial breast cancer cases compared with ancestry-matched population controls. The truncated PALB2 protein caused by this mutation retained little BRCA2-binding capacity and was deficient in homologous recombination and crosslink repair. Further screening of c.1592delT in unselected breast cancer individuals revealed a roughly fourfold enrichment of this mutation in patients compared with controls. Most of the mutation-positive unselected cases had a familial pattern of disease development. In addition, one multigenerational prostate cancer family that segregated the c.1592delT truncation allele was observed. These results indicate that PALB2 is a breast cancer susceptibility gene that, in a suitably mutant form, may also contribute to familial prostate cancer development.

Evaluation of the role of Finnish ataxia-telangiectasia mutations in hereditary predisposition to breast cancer.

Biallelic mutations in the ataxia-telangiectasia mutated (ATM) gene result in ataxia-telangiectasia (A-T). Studies on A-T families have shown that obligate female carriers have increased risk of developing breast cancer. Here we have evaluated the role of known Finnish ATM germ line mutations as possible breast cancer predisposing alleles outside A-T families by analyzing their prevalence in large cohorts of familial and unselected breast cancer cases. Of seven different alterations, two were observed in the studied breast cancer material. ATM 6903insA (causing protein truncation) was seen in 3/541 familial and 5/1124 unselected cases, but not among healthy population controls (0/1107). 7570G>C (Ala2524Pro) occurred in 1/541 familial and 2/1124 unselected cases compared with 1/1107 in controls. Additionally, 8734A>G (Arg2912Gly) associated previously with breast cancer susceptibility and suggested to be causative also for A-T was detected in 2/541 of familial cases, but not in unselected cases (0/1124) or controls (0/1107). In total, heterozygous ATM mutation carriers were observed in 6/541 familial [P = 0.006, odds ratio (OR) 12.4, 95% confidence interval (CI) 1.5-103.3) and 7/1124 unselected cases (P = 0.07, OR 6.9, 95% CI 0.9-56.4), compared with 1/1107 in controls, suggesting an apparent yet overall limited contribution to predisposition to cancer. The current results also provided evidence for founder effects in the geographical distribution of these mutations. Interestingly, results from functional analysis of the breast cancer-associated ATM mutations indicated that cancer susceptibility is not restricted to mutations with dominant-negative effect on kinase activity, displayed only by 7570G>C, whereas 8734A>G showed only a partial defect in the phosphorylation of ATM substrates, and 6903insA seemed to be a null allele.

Large genomic BRCA2 rearrangements and male breast cancer.

BACKGROUND: Germ-line mutations of the BRCA2 gene are the highest known risk factors for male breast cancer (MBC). Mutations in BRCA2 are mainly point mutations in contrast to BRCA1 in which large genomic rearrangements are quite common. In recent literature, however, genomic alterations of BRCA2 have been linked especially to male breast cancer families. We wanted to screen large genomic deletions and duplications of BRCA2 among Finnish male breast cancer patients. METHODS: We used multiplex ligation-dependent probe amplification (MLPA) to detect large genomic rearrangements in the BRCA2 gene among 36 unselected Finnish male breast cancer patients previously tested and found negative for Finnish BRCA1 and BRCA2 founder mutations. RESULTS: No genomic mutations of BRCA2 nor CHEK2*1100delC point mutations, also included in the assay, were found in this study. CONCLUSION: Large genomic BRCA2 rearrangements were not found among our 36 Finnish male breast cancer patients. Screening of large BRCA2 rearrangements is not likely to be advantageous in Finland.

BARD1 variants Cys557Ser and Val507Met in breast cancer predisposition.

BARD1 (BRCA1-associated RING-domain 1) is a tumor suppressor whose protein product interacts with BRCA1, and in which rare somatic and germline mutations have been reported in breast, uterine, and endometrial cancers. We aimed to evaluate whether there are BARD1 genetic variants that contribute to breast cancer risk by screening the gene for germline alterations in 45 Finnish familial breast cancer patients and in seven patients with both breast and ovarian cancer. Two of the missense alterations identified (Cys557Ser and Val507Met) were recently suggested to associate with an increased breast cancer risk. We also analyzed these variants in large and independent series of familial and unselected breast cancer patients and healthy controls. No clearly deleterious mutations were detected in the initial mutation screening. No association of the Cys557Ser and breast cancer risk was observed as the variant was found altogether in 1.4% (16/1181) of familial and 2.2% (34/1565) of unselected breast cancer patients, and in 2.5% (27/1083) of healthy controls. The frequency of the Val-allele of the Val507Met variant was modestly higher among breast cancer patients than among healthy controls, although the difference did not reach statistical significance. No statistically significant association of the Cys557Ser or Val507Met variants with any clinicopathologic parameters was observed. These results suggest that the contribution of the BARD1 germline variants to breast cancer predisposition is very limited, and that neither Cys557Ser nor Val507Met have an effect on familial breast cancer susceptibility.

Hemochromatosis gene mutations among Finnish male breast and prostate cancer patients.

Hereditary hemochromatosis (HH), the most common genetic disease in northern Europeans, is an autosomal recessive disorder of iron metabolism. The association between hepatocellular carcinoma and HFE homozygosity is well documented, but recently HFE hetero- and homozygosity has also been linked to nonhepatocellular malignancies, including female breast cancer. We hypothesized that C282Y and H63D mutations in the HFE gene could contribute to male breast cancer (MBC) and prostate cancer (PC) susceptibility at the population level in Finland. We screened the 2 major HFE mutations, H63D and C282Y, from 116 MBC cases diagnosed in Finland between 1967 and 1996, 843 consecutive unselected PC cases diagnosed at the Pirkanmaa Hospital District between 1999 and 2001 and 480 anonymous blood donor controls by minisequencing. Our results indicate that the frequencies of the HFE mutations do not significantly differ between MBC and PC patients and the population-based controls. No significantly altered risks for MBC or PC among carriers of the 2 variants were observed. However, HFE mutations were seen twice as often among carriers of a common BRCA2 mutation 9346(-2)A-->G compared with the rest of the MBC cases, indicating that HFE may be an MBC risk modifier gene among BRCA2 mutation carriers. In conclusion, our results indicate a minor role for the HFE mutations C282Y and H63D in the causation of MBC and PC, but carriers of both BRCA2 9346(-2)A-->G and an HFE mutation may be at an increased risk.

Search for large genomic alterations of the BRCA1 gene in a Finnish population.

Mutations in the BRCA1 and BRCA2 genes are known to predispose to breast cancer. In Finland, however, only 21% of all breast cancer families have mutations in these genes. Recent studies have shown that large genomic alterations of BRCA1 are common in many countries. Because such alterations will be missed in conventional mutation screening strategies, we decided to screen Finnish breast and ovarian cancer families for genomic alterations by using a multiplex polymerase chain reaction method. The most characteristic features of BRCA1-related breast cancer were used to select patients, namely (1) both breast and ovarian cancer in the family (48 patients), (2) four or more breast cancers in family (22 patients), or (3) young age (< or =40 years) of onset (58 patients). A total of 128 patients were included in the study. All exons of BRCA1 were analyzed but no alterations were found. This study excludes the frequent occurrence of large genomic alterations in the BRCA1 gene in Finland. Here, again, Finland differs from other countries with a mixed population structure. Our results are in agreement with the common hypothesis that there are still unknown breast cancer susceptibility gene(s) that are responsible for breast cancer predisposition.

Immunohistochemical expression of DNA repair proteins in familial breast cancer differentiate BRCA2-associated tumors.

PURPOSE: Morphologic and immunohistochemical studies of familial breast cancers have identified specific characteristics associated with BRCA1 mutation-associated tumors when compared with BRCA2 and non-BRCA1/2 tumors, but have not identified differences between BRCA2 and non-BRCA1/2 tumors. Because BRCA1 and BRCA2 genes participate in the DNA repair pathway, we have performed an immunohistochemical study with markers related to this pathway to establish the profile of the three groups. MATERIALS AND METHODS: We have studied two tissue microarrays that include 103 familial and 104 sporadic breast tumors, with a panel of DNA repair markers including ATM, CHEK2, RAD51, RAD50, XRCC3, and proliferating cell nuclear antigen. RESULTS: We found more frequent expression of CHEK2 in BRCA1 and BRCA2 tumors than in non-BRCA1/2 and sporadic tumors. We found absence of nuclear expression and presence of cytoplasmic expression of RAD51 in BRCA2 tumors that differentiate them from other familial tumors. We validated these results with a new series of patient cases. The final study with 253 familial patient cases (74 BRCA1, 71 BRCA2, 108 non-BRCA1/2), and 288 sporadic patient cases, has allowed us to confirm our preliminary results. Because BRCA2 tumors present a specific immunohistochemical profile for RAD51 and CHEK2 markers that is different from non-BRCA1/2 tumors, we have built a multivariate model with these markers that distinguish both tumors with an estimated probability of at least 76%. CONCLUSION: Our results suggest that BRCA2 tumors demonstrate more cytoplasmic and less nuclear RAD51 staining, and increased CHEK2 staining. This pattern may distinguish BRCA2 from familial non-BRCA1/2 tumors.

Matrix metalloproteinase3 and 9 gene promoter polymorphisms: joint action of two loci as a risk factor for coronary artery complicated plaques.

OBJECTIVE: Matrix metalloproteinases 3 and 9 (MMP3 and MMP9) are present in atherosclerotic plaques and co-operate in the degradation of the fibrous cap of the atheroma, leading to fissuring and ultimately to acute coronary thrombosis. The functional genetic polymorphisms in the promoters of MMP3 and MMP9, which lead to low- and high-transcription activity genotypes, have been shown to be associated with myocardial infarction and angiographically measured atherosclerosis individually, whereas their effects in combination are not yet known. In order to assess the two disease loci simultaneously, we investigated the association of combined low and high promoter activity genotypes with different types of coronary lesions in an autopsy cohort of 300 Caucasian males aged 33-69 years (Helsinki Sudden Death Study). METHODS: Genotyping at these loci was performed by PCR, restriction enzyme digestion and minisequencing, and areas of the coronary wall covered with atherosclerotic lesions were measured using computer-assisted morphometry. RESULTS: In analysis of covariance (ANCOVA) with age, body mass index, hypertension, diabetes, smoking and alcohol consumption as covariates, a significant interaction between the MMP3 and MMP9 genotypes was observed on area of complicated lesions (P=0.012). Men with high promoter activity genotypes for both loci had, on average, more than two times larger area of complicated lesions (250%) compared with those men who had low promoter activity genotypes (P=0.008), but these loci showed no association with myocardial infarction. CONCLUSIONS: The joint action of two susceptibility loci, rather than single MMP genes alone, and the particular combination of MMP3 and MMP9 genotypes present at these loci may contribute to heterogeneity in the presentation of atherosclerosis.

Correlation of CHEK2 protein expression and c.1100delC mutation status with tumor characteristics among unselected breast cancer patients.

The CHEK2 kinase is a tumor suppressor whose activation in response to DNA double-strand breaks contributes to cell cycle arrest or apoptosis. The c.1100delC mutation is associated with familial breast cancer, and tumors from mutation carriers show reduced or absent CHEK2 protein expression. We have here studied CHEK2 protein expression by immunohistochemistry on a tissue microarray of 611 unselected breast tumors and also evaluated the tumor characteristics among 1,297 unselected breast cancer patients defined for the c.1100delC germ line mutation status (2.5% carrier frequency). CHEK2 protein expression was reduced in 21.1% of the unselected breast cancers studied. Tumors with reduced CHEK2 expression had more often larger primary tumor size (pT3-4; nominal significance p = 0.002) compared to tumors with normal staining. A similar trend for larger tumor size was seen among the 37 breast tumors from c.1100delC germ line mutation carriers. Tumors from c.1100delC mutation carriers were of higher grade than those of noncarriers (nominal significance p = 0.02). The c.1100delC germ line mutation also associated strongly with bilateral breast cancer. No significant correlation was seen between CHEK2 status and hormone receptor status, histology, lymph node status, or overall survival.

BRCA2 mutations in 154 finnish male breast cancer patients.

The etiology and pathogenesis of male breast cancer (MBC) are poorly known. This is due to the fact that the disease is rare, and large-scale genetic epidemiologic studies have been difficult to carry out. Here, we studied the frequency of eight recurrent Finnish BRCA2 founder mutations in a large cohort of 154 MBC patients (65% diagnosed in Finland from 1967 to 1996). Founder mutations were detected in 10 patients (6.5%), eight of whom carried the 9346(-2) A>G mutation. Two novel mutations (4075 delGT and 5808 del5) were discovered in a screening of the entire BRCA2 coding region in 34 samples. However, these mutations were not found in the rest of the 120 patients studied. Patients with positive family history of breast and/or ovarian cancer were often BRCA2 mutation carriers (44%), whereas those with no family history showed a low frequency of involvement (3.6%; P < .0001). Finally, we found only one Finnish MBC patient with 999 del5, the most common founder mutation in Finnish female breast cancer (FBC) patients, and one that explains most of the hereditary FBC and MBC cases in Iceland. The variation in BRCA2 mutation spectrum between Finnish MBC patients and FBC patients in Finland and breast cancer patients in Iceland suggests that modifying genetic and environmental factors may significantly influence the penetrance of MBC and FBC in individuals carrying germline BRCA2 mutations in some populations.

Frequent amplification and overexpression of CCND1 in male breast cancer.

Genetic events underlying the pathogenesis of breast cancer have been studied extensively and several clinically significant markers have been identified. For example, amplification and overexpression of the ERBB2 oncogene is associated with poor prognosis in breast cancer and ERBB2 serves as a target for antibody-based therapy. Current knowledge on the pathogenesis of male breast cancer (MBC) is limited. The purpose of our study was to investigate the potential relevance of a series of genes known to be amplified in female breast cancer (FBC) in a the development and pathogenesis of MBC. To this end, we applied fluorescence in situ hybridization and immunohistochemistry to the analysis of 128 breast tumors from males. Amplification of ERBB2, MYC, PPM1D and ZNF217 was detected rarely (1-2% of tumors) indicating a considerably lower amplification frequency than in FBC. CCND1 amplification was observed in 12% of cases, being in good concordance with findings from FBC. In addition, CCND1 overexpression was detected in 63% of tumors and was associated with ER positivity (p < 0.0001). Our results indicate distinct differences in the genetic basis of MBC and FBC and suggest that marked differences exist in the pathogenesis of these diseases. The lack of ERBB2 involvement was especially unexpected and implies that ERBB2-targeted therapies are unlikely to be beneficial in MBC. Furthermore, the high frequency of hormone receptor positivity and the association between ER positivity and CCND1 overexpression supports the notion that hormonal regulation is likely to be essential for the development of MBC.

CHEK2 variant I157T may be associated with increased breast cancer risk.

Cell cycle checkpoint kinase 2 (CHEK2) is a transducer of cellular responses to DNA damage. The CHEK2 1100delC has previously been shown to be a low-penetrance breast cancer susceptibility allele. We have evaluated the role of another CHEK2 variant, I157T in the FHA domain of the gene, for association with breast cancer. I157T was found at a significantly higher frequency in the population-based series of breast cancer patients (77/1035, 7.4%, odds ratio [OR] = 1.43, 95% confidence interval [CI] = 1.06-1.95, p = 0.021) than among population controls (100/1885, 5.3%). The frequency in the familial breast cancer patients was not elevated (28/507, 5.5%, OR = 1.04, 95% CI = 0.68-1.61). The I157T protein, that undermines cellular responses to ionizing radiation and shows deficiency in substrate recognition in vivo, was expressed at normal level in tumor tissues as well as in cultured cells. The I157T protein was stable and it dimerized with the wild-type CHEK2 co-expressed in human cells. These functional properties of the I157T protein suggest that this variant may have negative effect on the pool of normal CHEK2 protein in heterozygous carrier cells by formation of heterodimers with wild-type CHEK2. The I157T variant may be associated with breast cancer risk, but the risk is lower than for 1100delC.

CHEK2 1100delC is not a risk factor for male breast cancer population.

Genetic risk factors for male breast cancer (MBC) are poorly understood. High penetrance genes such as BRCA1 or BRCA2 account for only a small proportion of the disease. A 1100delC mutation in CHEK2 (previously known as CHK2), a cell-cycle checkpoint kinase, has been implicated in predisposition of Li-Fraumeni syndrome (LFS) and breast cancer in families suggestive of LFS. This 1100delC mutation has also been shown to confer a 2-fold increase of breast cancer risk in women and a 10-fold increase of risk in men. It was estimated to account for 1% of breast cancers in women and as much as 9% of breast cancers in men at the population level based on analysis of breast cancer families without BRCA1 or BRCA2 mutations. We wanted to evaluate the significance of CHEK2 1100delC in predisposition to MBC by assessing its frequency in a population-based material of 114 Finnish MBC patients. Two patients (1.8%) carried the 1100delC mutation. The mutation frequency among MBC cases was similar to that seen in population controls (26/1885, 1.4%). Our results indicate that CHEK2 1100delC variant does not substantially increase the risk of male breast cancer at the population level. We cannot exclude the fact that a small fraction of hereditary, family-positive male breast cancers could be attributable to CHEK2 mutations.

Androgen receptor gene alterations in Finnish male breast cancer.

Mutations in the androgen receptor (AR) gene have been suggested to predispose to male breast cancer (MBC). Studies on MBC patients have not been based on the mutation screening of the entire coding region of the AR and the number of subjects has been small. Therefore, some AR gene alterations may have remained undetected. In the present study, we have comprehensively screened the entire coding region of the AR gene for mutations and also studied the role of AR CAG and GGC repeat lengths as risk factors for MBC in a cohort of 32 Finnish MBC patients. To estimate the possible involvement of the prostate cancer predisposing AR Arg726Leu germ-line mutation in MBC, this mutation was tested in 117 MBC patients. No germ-line mutations were found and the CAG and GGC repeat lengths were similar among MBC cases as among Scandinavian population. Our data indicate that the AR gene does not substantially contribute to MBC predisposition.

Coronary artery calcification is related to functional polymorphism of matrix metalloproteinase 3: the Helsinki Sudden Death Study.

Matrix metalloproteinase 3 (MMP3) is expressed in human coronary atherosclerotic lesions and is known to be involved in degradation of the plaque and to be co-localized with calcium and fibrin deposits in advanced lesions, indicating a possible role of MMP3 in arterial calcification. The MMP3 gene promoter polymorphism leads to low promoter activity 6A6A, intermediate promoter activity 5A6A and high promoter activity 5A5A genotypes. To determine whether these genotypes predict the extent of atherosclerosis we investigated their association with different types of coronary lesions in an autopsy series of 300 middle-aged white Finnish men (aged 35-69 years) from the Helsinki Sudden Death Study (HSDS). Areas of the coronary wall covered with different atherosclerotic lesions were measured and MMP3 genotypes were determined by PCR and minisequencing. In men >/=53 years the mean area of calcified lesion in the most severely affected coronary artery was significantly associated with the MMP3 genotype (P=0.029). Subjects with high promoter activity genotypes had on average larger calcified lesion areas than those with the low-activity genotype. The MMP3 genotype (P=0.025) persisted as an independent predictor of mean calcified lesion area after stepwise adjustment for age, BMI, hypertension, diabetes, number of affected vessels and smoking. These data provide evidence that the proposed effect of MMP3 in the process of atherogenesis may be modified by the MMP3 genotype.

A CHEK2 genetic variant contributing to a substantial fraction of familial breast cancer.

CHEK2 (previously known as "CHK2") is a cell-cycle-checkpoint kinase that phosphorylates p53 and BRCA1 in response to DNA damage. A protein-truncating mutation, 1100delC in exon 10, which abolishes the kinase function of CHEK2, has been found in families with Li-Fraumeni syndrome (LFS) and in those with a cancer phenotype that is suggestive of LFS, including breast cancer. In the present study, we found that the frequency of 1100delC was 2.0% among an unselected population-based cohort of 1,035 patients with breast cancer. This was slightly, but not significantly (P=.182), higher than the 1.4% frequency found among 1,885 population control subjects. However, a significantly elevated frequency was found among those 358 patients with a positive family history (11/358 [3.1%]; odds ratio [OR] 2.27; 95% confidence interval [CI] 1.11-4.63; P=.021, compared with population controls). Furthermore, patients with bilateral breast cancer were sixfold more likely to be 1100delC carriers than were patients with unilateral cancer (95% CI 1.87-20.32; P=.007). Analysis of the 1100delC variant in an independent set of 507 patients with familial breast cancer with no BRCA1 and BRCA2 mutations confirmed a significantly elevated frequency of 1100delC (28/507 [5.5%]; OR 4.2; 95% CI 2.4-7.2; P=.0002), compared with controls, with a high frequency also seen in patients with only a single affected first-degree relative (18/291 [6.2%]). Finally, tissue microarray analysis indicated that breast tumors from patients with 1100delC mutations show reduced CHEK2 immunostaining. The results suggest that CHEK2 acts as a low-penetrance tumor-suppressor gene in breast cancer and that it makes a significant contribution to familial clustering of breast cancer-including families with only two affected relatives, which are more common than families that include larger numbers of affected women.