Identification of 33 candidate oncogenes by screening for base-specific mutations.

BACKGROUND: Genes with recurrent codon-specific somatic mutations are likely drivers of tumorigenesis and potential therapeutic targets. Hypermutable cancers may represent a sensitive system for generation and selection of oncogenic mutations. METHODS: We utilised exome-sequencing data on 25 sporadic microsatellite-instable (MSI) colorectal cancers (CRCs) and searched for base-specific somatic mutation hotspots. RESULTS: We identified novel mutation hotspots in 33 genes. Fourteen genes displayed mutations in the validation set of 254 MSI CRCs: ANTXR1, MORC2, CEP135, CRYBB1, GALNT9, KRT82, PI15, SLC36A1, CNTF, GLDC, MBTPS1, OR9Q2, R3HDM1 and TTPAL. A database search found examples of the hotspot mutations in multiple cancer types. CONCLUSIONS: This work reveals a variety of new recurrent candidate oncogene mutations to be further scrutinised as potential therapeutic targets.

Mutation analysis of components of the Mediator kinase module in MED12 mutation-negative uterine leiomyomas.

BACKGROUND: Kinase module of Mediator complex ('CDK8 submodule') consists of four subunits: CDK8, Cyclin C, MED12, and MED13. Recently, we reported recurrent MED12 mutations in 70% of uterine leiomyomas. The aim of this study was to analyse whether mutations in other components of the module contribute to the development of these lesions. METHODS: Mutation screening of altogether 70 MED12 mutation-negative uterine leiomyomas was carried out by direct sequencing. RESULTS: None of the tumours displayed somatic mutations in the coding regions of CDK8/CDK19, CCNC, or MED13. CONCLUSIONS: Mutations in CDK8/CDK19, CCNC, and MED13 do not frequently contribute to genesis of uterine leiomyomas.

Diagnostic cancer genome sequencing and the contribution of germline variants.

Whole-genome sequencing (WGS) is revolutionizing medical research and has the potential to serve as a powerful and cost-effective diagnostic tool in the management of cancer. We review the progress to date in the use of WGS to reveal how germline variants and mutations may be associated with cancer. We use colorectal cancer as an example of how the current level of knowledge can be translated into predictions of predisposition. We also address challenges in the clinical implementation of the variants in germline DNA identified through cancer genome sequencing. We call for the international development of standards to facilitate the clinical use of germline information arising from diagnostic cancer genome sequencing.

Somatic MED12 mutations in uterine leiomyosarcoma and colorectal cancer.

BACKGROUND: Mediator complex participates in transcriptional regulation by connecting regulatory DNA sequences to the RNA polymerase II initiation complex. Recently, we discovered through exome sequencing that as many as 70% of uterine leiomyomas harbour specific mutations in exon 2 of mediator complex subunit 12 (MED12). In this work, we examined the role of MED12 exon 2 mutations in other tumour types. METHODS: The frequency of MED12 exon 2 mutations was analysed in altogether 1158 tumours by direct sequencing. The tumour spectrum included mesenchymal tumours (extrauterine leiomyomas, endometrial polyps, lipomas, uterine leiomyosarcomas, other sarcomas, gastro-intestinal stromal tumours), hormone-dependent tumours (breast and ovarian cancers), haematological malignancies (acute myeloid leukaemias, acute lymphoid leukaemias, myeloproliferative neoplasms), and tumours associated with abnormal Wnt-signalling (colorectal cancers (CRC)). RESULTS: Five somatic alterations were observed: three in uterine leiomyosarcomas (3/41, 7%; Gly44Ser, Ala38_Leu39ins7, Glu35_Leu36delinsVal), and two in CRC (2/392, 0.5%; Gly44Cys, Ala67Val). CONCLUSION: Somatic MED12 exon 2 mutations were observed in uterine leiomyosarcomas, suggesting that a subgroup of these malignant tumours may develop from a leiomyoma precursor. Mutations in CRC samples indicate that MED12 may, albeit rarely, contribute to CRC tumorigenesis.

delGA (rs67491583) variant and colorectal cancer risk in an indigenous African population.

BACKGROUND: A recent study showed a higher frequency of GA deletion at rs67491583 in African American colorectal cancer (CRC) patients compared to controls, suggesting a likely contribution of this allele to racial disparity in CRC risk predisposition. We conducted a pilot study in an indigenous African population to evaluate this potential CRC risk variant. METHODS: We collected epidemiological data and biological specimen from consenting consecutive CRC cases and controls presenting at the Oncology Clinic of University College Hospital, Ibadan from 2001 to 2007. We examined germline DNA for delGA by PCR-amplification of two overlapping fragments using standard primers. The products were directly sequenced using Applied Biosystems BigDye v3.1 sequencing chemistry and AB 13730 automatic DNA sequencer. RESULTS: There were 45 cases and 45 controls of which genotyping was successful in 39 cases and 38 controls. There were 5 heterozygous and 2 homozygous GA deletions with frequency of 11.54% (9/78) among cases whereas there were 8 heterozygous and 1 homozygous GA deletions among controls with frequency of 13.15% (10/76). (p= 0.79, OR 0.88, 95% CI 0.34-2.28). CONCLUSION: This study suggests that there is no association between the delGA (rs67491583) variant and CRC risk in this indigenous African population. However our sample size was small and the participants were not ethnically homogenous. Further studies are required to evaluate this marker in African CRC.

A large-scale meta-analysis to refine colorectal cancer risk estimates associated with MUTYH variants.

BACKGROUND: defective DNA repair has a causal role in hereditary colorectal cancer (CRC). Defects in the base excision repair gene MUTYH are responsible for MUTYH-associated polyposis and CRC predisposition as an autosomal recessive trait. Numerous reports have suggested MUTYH mono-allelic variants to be low penetrance risk alleles. We report a large collaborative meta-analysis to assess and refine CRC risk estimates associated with bi-allelic and mono-allelic MUTYH variants and investigate age and sex influence on risk. METHODS: MUTYH genotype data were included from 20 565 cases and 15 524 controls. Three logistic regression models were tested: a crude model; adjusted for age and sex; adjusted for age, sex and study. RESULTS: all three models produced very similar results. MUTYH bi-allelic carriers demonstrated a 28-fold increase in risk (95% confidence interval (CI): 6.95-115). Significant bi-allelic effects were also observed for G396D and Y179C/G396D compound heterozygotes and a marginal mono-allelic effect for variant Y179C (odds ratio (OR)=1.34; 95% CI: 1.00-1.80). A pooled meta-analysis of all published and unpublished datasets submitted showed bi-allelic effects for MUTYH, G396D and Y179C (OR=10.8, 95% CI: 5.02-23.2; OR=6.47, 95% CI: 2.33-18.0; OR=3.35, 95% CI: 1.14-9.89) and marginal mono-allelic effect for variants MUTYH (OR=1.16, 95% CI: 1.00-1.34) and Y179C alone (OR=1.34, 95% CI: 1.01-1.77). CONCLUSIONS: overall, this large study refines estimates of disease risk associated with mono-allelic and bi-allelic MUTYH carriers.

COGENT (COlorectal cancer GENeTics): an international consortium to study the role of polymorphic variation on the risk of colorectal cancer.

It is now recognised that a part of the inherited risk of colorectal cancer (CRC) can be explained by the co-inheritance of low-penetrance genetic variants. The accumulated experience to date in identifying these variants has served to highlight difficulties in conducting statistically and methodologically rigorous studies and follow-up analyses. The COGENT (COlorectal cancer GENeTics) consortium includes 20 research groups in Europe, Australia, the Americas, China and Japan. The overarching goal of COGENT is to identify and characterise low-penetrance susceptibility variants for CRC through association-based analyses. In this study, we review the rationale for identifying low-penetrance variants for CRC and our proposed strategy for establishing COGENT.

Aryl hydrocarbon receptor interacting protein mutations seem not to associate with familial non-medullary thyroid cancer.

BACKGROUND: Over 95% of all thyroid malignancies are non-medullary thyroid carcinomas (NMTC). Familial NMTC are more aggressive and mortality is higher as compared with sporadic carcinomas. Known genetic factors do not explain all familial NMTC. Recently, thyroid disorders have been observed in families with germline mutations in aryl hydrocarbon receptor interacting protein (AIP) but, due to frequent occurrence of these conditions in the population, the significance of this co-occurrence is not clear. AIM, SUBJECTS AND METHODS: To examine whether AIP is involved in familial NMTC, we performed AIP mutation screening in 93 familial NMTC cases. In addition, the AIP status was studied in one follicular thyroid adenoma patient with a known AIP mutation from an additional cohort. RESULTS: No potentially pathogenic changes were identified, but two likely rare polymorphisms were detected. AIP mutation-positive patient's follicular thyroid adenoma showed no loss of heterozygosity or lack of immunohistochemical AIP staining. CONCLUSION: Our study indicates that germline AIP mutations are rare or do not exist in familial NMTC.

Mutation analysis of MEN1, HRPT2, CASR, CDKN1B, and AIP genes in primary hyperparathyroidism patients with features of genetic predisposition.

OBJECTIVE: Primary hyperparathyroidism (PHPT), a common endocrine condition, is usually caused by sporadically occurring parathyroid adenoma. A subset of patients carry germline mutations in genes such as MEN1 (multiple endocrine neoplasia type 1), HRPT2 (hyperparathyroidism 2), and CASR (calcium-sensing receptor) predisposing to syndromic forms of PHPT or familial isolated hyperparathyroidism (FIHP). Recently, germline mutations in two novel genes AIP (aryl hydrocarbon receptor-interacting protein) and CDKN1B (cyclin-dependent kinase inhibitor 1B) have been found to be associated with endocrine tumors. The purpose of this study was to evaluate the role of MEN1, HRPT2, CASR, AIP, and CDKN1B genes in PHPT patients with clinical features suggestive of genetic predisposition. PATIENTS AND DESIGN: Medical records of patients treated for PHPT from 1974 to 2001 at Oulu University Hospital were reviewed. Patients with multiglandular or recurrent/persistent disease, other MEN1- related manifestations, aged 40 yr or younger at onset or with a family history of PHPT/MEN1-related tumor were invited to the study. Twenty patients with previously diagnosed MEN1 were excluded. Participants were interviewed and blood samples obtained for biochemical screening and mutation analysis of MEN1, HRPT2, CASR, AIP, and CDKN1B. RESULTS: Of the 56 invited patients, 29 took part in the study. One patient was found to carry the c. 1356_1367del12 MEN1 founder mutation. Mutations in other genes were not detected. CONCLUSIONS: Apart from MEN1, mutations in other genes predisposing to PHPT seem to be rare or non-existing in Northern Finnish PHPT patients. No evidence was found for a role of AIP or CDKN1B in PHPT predisposition.

Downregulation of SRF-FOS-JUNB pathway in fumarate hydratase deficiency and in uterine leiomyomas.

Defects of metabolic enzymes result in a variety of manifestations not logically explained by the primary metabolic function. Dominant defects of fumarate hydratase (FH) result in predisposition to cutaneous and uterine leiomyomas, and renal cell cancer. FH is a metabolic enzyme of the tricarboxylic acid cycle, and its tumor-suppressor mechanism is not fully understood. We compared the consequences of FH deficiency and respiratory chain (RC) deficiency using global expression pattern of diploid primary fibroblasts. This approach utilized the information that RC defects do not seem to predispose to tumorigenesis, and the aim was to identify FH-specific signaling effects that might have relevance to tumor formation. These results were then compared to global expression patterns of FH-deficient and sporadic uterine leiomyoma data sets. We show here that FH-deficient fibroblasts share a common transcriptional fingerprint with FH-deficient and sporadic leiomyomas, highlighting the downregulation of serum response factor (SRF)-regulated transcripts, particularly the FOS-JUNB pathway. We confirmed the downregulation of this pathway at transcriptional and protein level. SRF has a fundamental function in the differentiation of smooth muscle progenitor cells, and its downregulation both in diploid FH-deficient primary fibroblasts and in leiomyomas suggests an early function in the mechanism of uterine leiomyoma formation in FH deficiency. Concordantly, the phosphorylated form of SRF, known to activate transcription, is undetectable in leiomyomas whereas clearly detected in several nuclei in the differentiated myometrium. A similar transcriptional SRF-pathway fingerprint in FH-deficient and sporadic leiomyomas emphasizes the potential importance of this pathway in primary events leading to leiomyomatosis.

Dysregulation of the transcription factors SOX4, CBFB and SMARCC1 correlates with outcome of colorectal cancer.

The aim of this study was to identify deregulated transcription factors (TFs) in colorectal cancer (CRC) and to evaluate their relation with the recurrence of stage II CRC and overall survival. Microarray-based transcript profiles of 20 normal mucosas and 424 CRC samples were used to identify 51 TFs displaying differential transcript levels between normal mucosa and CRC. For a subset of these we provide in vitro evidence that deregulation of the Wnt signalling pathway can lead to the alterations observed in tissues. Furthermore, in two independent cohorts of microsatellite-stable stage II cancers we found that high SOX4 transcript levels correlated with recurrence (HR 2.7; 95% CI, 1.2-6.0; P=0.01). Analyses of approximately 1000 stage I-III adenocarcinomas, by immunohistochemistry, revealed that patients with tumours displaying high levels of CBFB and SMARCC1 proteins had a significantly better overall survival rate (P=0.0001 and P=0.0275, respectively) than patients with low levels. Multivariate analyses revealed that a high CBFB protein level was an independent predictor of survival. In conclusion, several of the identified TFs seem to be involved in the progression of CRC.

No evidence of somatic aryl hydrocarbon receptor interacting protein mutations in sporadic endocrine neoplasia.

Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene were recently observed in patients with pituitary adenoma predisposition (PAP). Though AIP mutation-positive individuals with prolactin-, mixed growth hormone/prolactin-, and ACTH-producing pituitary adenomas as well as non-secreting pituitary adenomas have been reported, most mutation-positive patients have had growth hormone-producing adenomas diagnosed at relatively young age. Pituitary adenomas are also component tumors of some familial endocrine neoplasia syndromes such as multiple endocrine neoplasia type 1 (MEN1) and Carney complex (CNC). Genes underlying MEN1 and CNC are rarely mutated in sporadic pituitary adenomas, but more often in other lesions contributing to these two syndromes. Thus far, the occurrence of somatic AIP mutations has not been studied in endocrine tumors other than pituitary adenomas. Here, we have analyzed 32 pituitary adenomas and 79 other tumors of the endocrine system for somatic AIP mutations by direct sequencing. No somatic mutations were identified. However, two out of nine patients with prolactin-producing adenoma were shown to harbor a Finnish founder mutation (Q14X) with a complete loss of the wild-type allele in the tumors. These results are in agreement with previous studies in that prolactin-producing adenomas are component tumors in PAP. The data also support the previous finding that somatic AIP mutations are not common in pituitary adenomas and suggest that such mutations are rare in other endocrine tumors as well.

Blood-derived gene-expression profiling in unravelling susceptibility to recessive disease.

Identification of new disease predisposition genes with chip-based technologies typically requires extensive financial and sample resources. We have recently shown that combining peripheral blood genome and transcriptome (BGT) information in highly selected materials can be a successful low-cost approach to unravelling dominant tumour susceptibility. In this study, we extended our investigations to recessively inherited tumour predisposition, and identified a homozygous germline mutation in the damage-specific DNA binding protein 2 (DDB2) gene in a patient with several facial tumours, for which doctors had been unable to provide a diagnosis. Our results provide proof of principle that BGT is a powerful approach for both dominant and recessive genes. In addition to tumour susceptibility, the method may be useful in characterising genetic defects underlying other disease phenotypes.

Direct contact in inviting high-risk members of hereditary colon cancer families to genetic counselling and DNA testing.

BACKGROUND: Identification of hereditary predisposition to cancer has limited significance if not followed by efficient cancer prevention in the family. Probands are traditionally left to inform their relatives about the increased risk, but distant relatives may remain uninformed. An approach to contacting directly at-risk persons assumed to be unaware of their increased cancer risk was taken. With cancer prevention as the ultimate goal, the study was aimed at investigating attitudes towards and psychosocial consequences of this novel strategy. METHODS: In families with hereditary non-polyposis colorectal cancer (Lynch syndrome), 286 healthy adult relatives with a 50% risk of a predisposing mutation were contacted by letter. Of these, 112 participated in counselling and predictive testing. Baseline information and information obtained 1 month after the test for 73 respondents were compared with 299 corresponding subjects, approached via the proband (family-mediated approach in our previous study) in these families. RESULTS: After the contact letter, 51% consented to the study. Of these, 92% approved of the direct contact and 33% had tried to seek information. In 34% of the mutation carriers, neoplasia was identified in the first post-test colonoscopy. Although post-test fear of cancer increased among the mutation carriers and decreased among noncarriers, almost all participants were satisfied with their decision to participate, independently of their test results, parallel to the family-mediated approach. CONCLUSION: In this large-scale study, relatives in cancer families were actively contacted to inform them of the condition and genetic counselling. Their attitudes were encouraging, and the psychosocial consequences were similar to the family-mediated approach. Our results suggest the appropriateness of direct contact as an alternative method of contact in cases of life-threatening treatable disease.

Differential expression of DHHC9 in microsatellite stable and instable human colorectal cancer subgroups.

Microarray analysis on pooled samples has previously identified ZDHHC9 (DHHC9) to be upregulated in colon adenocarcinoma compared to normal colon mucosa. Analyses of 168 samples from proximal and distal adenocarcinomas using U133plus2.0 microarrays validated these findings, showing a significant two-fold (log 2) upregulation of DHHC9 transcript (P<10(-6)). The upregulation was more striking in microsatellite stable (MSS), than in microsatellite instable (MSI), tumours. Genes known to interact with DHHC9 as H-Ras or N-Ras did not show expression differences between MSS and MSI. Immunohistochemical analysis was performed on 60 colon adenocarcinomas, previously analysed on microarrays, as well as on tissue microarrays with 40 stage I-IV tumours and 46 tumours from different organ sites. DHHC9 protein was strongly expressed in MSS compared to MSI tumours, readily detectable in premalignant lesions, compared to the rare expression seen in normal mucosa. DHHC9 was specific for tumours of the gastrointestinal tract and localised to the Golgi apparatus, in vitro and in vivo. Overexpression of DHHC9 decreased the proliferation of SW480 and CaCo2 MSS cell lines significantly. In conclusion, DHHC9 is a gastrointestinal-related protein highly expressed in MSS colon tumours. The palmitoyl transferase activity, modifying N-Ras and H-Ras, suggests DHHC9 as a target for anticancer drug design.

Mutation analysis of aryl hydrocarbon receptor interacting protein (AIP) gene in colorectal, breast, and prostate cancers.

Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene were recently identified in individuals with pituitary adenoma predisposition (PAP). These patients have prolactin (PRL) or growth hormone (GH) oversecreting pituitary adenomas, the latter exhibiting acromegaly or gigantism. Loss-of-heterozygosity (LOH) analysis revealed that AIP is lost in PAP tumours, suggesting that it acts as a tumour-suppressor gene. Aryl hydrocarbon receptor interacting protein is involved in several pathways, but it is best characterised as a cytoplasmic partner of the aryl hydrocarbon receptor (AHR). To examine the possible role of AIP in the genesis of common cancers, we performed somatic mutation screening in a series of 373 colorectal cancers (CRCs), 82 breast cancers, and 44 prostate tumour samples. A missense R16H (47G>A) change was identified in two CRC samples, as well as in the respective normal tissues, but was absent in 209 healthy controls. The remaining findings were silent, previously unreported, changes of the coding, non-coding, or untranslated regions of AIP. These results suggest that somatic AIP mutations are not common in CRC, breast, and prostate cancers.

Serrated carcinomas form a subclass of colorectal cancer with distinct molecular basis.

Serrated colorectal carcinomas (CRCs) are morphologically different from conventional CRCs and have been proposed to follow a distinct pathway of CRC formation. Despite studies of single molecular events in this tumor type, the diagnosis of serrated CRC relies on morphology and the putative unique biological character of these tumors has not been established. Here we show that the gene expression profiling of 37 CRCs separated serrated and conventional CRCs into two distinct branches in unsupervised hierarchical clustering (P-value 7.8 x 10(-7)), and revealed 201 differentially expressed genes representing potential biomarkers for serrated CRC. Immunohistochemistry was utilized to verify the key findings in the 37 CRCs examined by expression profiling, and a separate validation set of 37 serrated and 86 conventional CRCs was examined to evaluate the candidate biomarkers in an extended sample material. Ephrin receptor B2, hypoxia-inducible factor 1-alpha and patched appeared as proteins important for genesis of serrated CRC. This study establishes serrated CRCs as a biologically distinct subclass of CRC and represents a step forward in the molecular classification of these cancers. The study also provides a platform to understand the molecular basis of serrated CRC and in long term may contribute to the development of specific treatment options for this tumor type.

No evidence for dual role of loss of heterozygosity in hereditary non-polyposis colorectal cancer.

Hereditary non-polyposis colorectal cancer (HNPCC) is caused by germline mutations in mismatch repair (MMR) genes, mostly MLH1 and MSH2. Somatic inactivation of the wild-type allele of the respective MMR gene is required for tumor development. Unexpectedly, a recent study utilizing DNA from paraffin-embedded tissue material detected frequent loss of the mutant MMR gene allele in HNPCC tumors. Dual role for loss of heterozygosity (LOH) was proposed. If somatic loss of the wild-type MMR gene allele had occurred through point mutation or promoter hypermethylation, frequent somatic deletions at the region of the MMR gene locus, perhaps targeting other relevant cancer genes, could quite commonly lead to loss of the mutant allele. To test this hypothesis, we studied a population-based series of 25 fresh-frozen HNPCC tumors with a germline mutation in MLH1 or MSH2 for LOH. Fourteen of the 25 tumors (56%) showed LOH at the respective locus, and all 14 losses targeted the wild-type allele (P=0.00006). These results strongly support the traditional two-hit model of HNPCC gene inactivation.

CHEK2 I157T associates with familial and sporadic colorectal cancer.

BACKGROUND: Recently, a functionally defective CHEK2 variant I157T has been proposed to associate with an increased risk of several types of cancer. We investigated the CHEK2 I157T variant for colorectal cancer (CRC) predisposition in a large population based study including a significant number of familial CRC cases. METHODS: We screened the CHEK2 I157T variant in a population based series of 1042 Finnish CRC patients using restriction fragment length polymorphism. Mutation status was studied for correlation with clinical characteristics and family history of CRC and other cancers. RESULTS: The frequency of CHEK2 I157T was significantly higher in CRC patients (7.8%, 76/972) than in healthy population controls (5.3%, 100/1885) (OR = 1.5, 95% CI 1.1 to 2.1, p = 0.008). The significant association of CHEK2 I157T with CRC was observed among patients with (10.4%, 14/135) and without (7.4%, 62/837) a family history of CRC (OR = 2.1, 95% CI 1.1 to 3.7, p = 0.01; OR = 1.4, 95% CI 1.0 to 2.0, p = 0.03; respectively). A trend towards higher variant frequency was also noted among patients with multiple primary tumours and a family history of any cancer. CONCLUSIONS: CHEK2 I157T associates with an increased risk of CRC: the association was observed both among familial and sporadic CRC patients. Furthermore, the higher frequency of I157T among patients with multiple primary tumours as well as those with a family history of any cancer supports a role for CHEK2 I157T as a susceptibility allele for multiple cancer types.

Nordic collaborative study of the BARD1 Cys557Ser allele in 3956 patients with cancer: enrichment in familial BRCA1/BRCA2 mutation-negative breast cancer but not in other malignancies.

BACKGROUND: BARD1 was originally identified as a BRCA1-interacting protein but has also been described in tumour-suppressive functions independent of BRCA1. Several studies have indicated that the BARD1 gene is a potential target for germline changes predisposing to breast and ovarian cancer. The C-terminal Cys557Ser change has previously been uncovered to associate with an increased risk of breast cancer and was recently shown to result in defective apoptotic activities. AIM AND METHODS: Conformation-sensitive gel electrophoresis, minisequencing, TaqMan assays, denaturing high-performance liquid chromatography analysis and DNA sequencing were used to investigate the prevalence of the Cys557Ser allele in a large Nordic case-control study cohort consisting of 2906 patients with breast or ovarian cancer, 734 with prostate cancer, 188 with colorectal cancer, 128 men with breast cancer, and 3591 controls from Finland, Iceland, Denmark, Sweden and Norway. RESULTS: The frequency of the BARD1 Cys557Ser variant seemed to increase among patients from families with breast or ovarian cancer lacking BRCA1 or BRCA2 mutations: a significant difference was obtained compared with controls (6.8% v 2.7%; p<0.001; odds ratio (OR) 2.6; 95% confidence interval (CI) 1.7 to 4.0) and with patients from BRCA1/BRCA2 mutation-positive families (6.8% v 2.2%; p = 0.01; OR 3.2; 95% CI 1.2 to 8.3). In contrast, no major association with male breast, ovarian, colorectal or prostate cancer was observed. Additionally, a novel BARD1 allele resulting in Ser558Pro was identified in familial breast cancer cases. CONCLUSION: These results provide further evidence that BARD1 Cys557Ser confers a slightly increased risk of breast cancer in women.