Modeling the evolution space of breakage fusion bridge cycles with a stochastic folding process.

Breakage-fusion-bridge cycles in cancer arise when a broken segment of DNA is duplicated and an end from each copy joined together. This structure then 'unfolds' into a new piece of palindromic DNA. This is one mechanism responsible for the localised amplicons observed in cancer genome data. Here we study the evolution space of breakage-fusion-bridge structures in detail. We firstly consider discrete representations of this space with 2-d trees to demonstrate that there are [Formula: see text] qualitatively distinct evolutions involving [Formula: see text] breakage-fusion-bridge cycles. Secondly we consider the stochastic nature of the process to show these evolutions are not equally likely, and also describe how amplicons become localized. Finally we highlight these methods by inferring the evolution of breakage-fusion-bridge cycles with data from primary tissue cancer samples.

Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts.

BACKGROUND: Myelodysplastic syndromes are a diverse and common group of chronic hematologic cancers. The identification of new genetic lesions could facilitate new diagnostic and therapeutic strategies. METHODS: We used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 9 patients with low-grade myelodysplasia. Targeted resequencing of the gene encoding RNA splicing factor 3B, subunit 1 (SF3B1), was also performed in a cohort of 2087 patients with myeloid or other cancers. RESULTS: We identified 64 point mutations in the 9 patients. Recurrent somatically acquired mutations were identified in SF3B1. Follow-up revealed SF3B1 mutations in 72 of 354 patients (20%) with myelodysplastic syndromes, with particularly high frequency among patients whose disease was characterized by ring sideroblasts (53 of 82 [65%]). The gene was also mutated in 1 to 5% of patients with a variety of other tumor types. The observed mutations were less deleterious than was expected on the basis of chance, suggesting that the mutated protein retains structural integrity with altered function. SF3B1 mutations were associated with down-regulation of key gene networks, including core mitochondrial pathways. Clinically, patients with SF3B1 mutations had fewer cytopenias and longer event-free survival than patients without SF3B1 mutations. CONCLUSIONS: Mutations in SF3B1 implicate abnormalities of messenger RNA splicing in the pathogenesis of myelodysplastic syndromes. (Funded by the Wellcome Trust and others.).

Familial male breast cancer is not linked to the BRCA1 locus on chromosome 17q.

Breast cancer in men is about a hundredfold less common than in women and this has hindered research into its genetic basis. We have examined 22 families with at least one case of male breast cancer for linkage to the hereditary breast and ovarian cancer locus, BRCA1, on chromosome 17q. We found strong evidence against linkage to BRCA1 (lod score-16.63) and the best estimate of the proportion of linked families was 0% (95% CI 0-18%). Our results indicate that there is a gene(s) other than BRCA1 which predisposes to early-onset breast cancer in women and which confers a higher risk of male breast cancer. Identification of additional pedigrees that include cases of male breast cancer may therefore facilitate the mapping and isolation of this gene.

A germline mutation in the androgen receptor gene in two brothers with breast cancer and Reifenstein syndrome.

Breast cancer in men is rare--among the risk factors that have been identified are a family history of breast cancer and evidence of androgen insufficiency. We report a family in which two brothers who both developed breast cancer had clinical and endocrinological evidence of androgen resistance. Sequence analysis revealed a mutation in the androgen receptor gene on the X chromosome, within the region encoding the DNA binding domain. This is the first report of a germline mutation in a member of the steroid/thyroid hormone receptor superfamily associated with the development of cancer.

Variation of risks of breast and ovarian cancer associated with different germline mutations of the BRCA2 gene.

The breast cancer susceptibility gene BRCA2 on chromosome 13q12-13 has recently been identified. Germline mutations of BRCA2 are predicted to account for approximately 35% of families with multiple case, early onset female breast cancer, and they are also associated with an increased risk of male breast cancer, ovarian cancer, prostate cancer and pancreatic cancer. Germline mutations of a second cancer susceptibility gene BRCA1 (ref. 5), are associated with a strong predisposition to ovarian cancer as well as female breast cancer. Recent studies have suggested that the phenotype in BRCA1 families with respect to the ratio of breast to ovarian cancer varies with the location of the BRCA1 mutation. To determine whether germline mutations in BRCA2 are associated with a similar variation in phenotypic risk, we have analysed the distribution of mutations in 25 families with multiple cases of breast and/or ovarian cancer ascertained in the United Kingdom and Eire. These mutations all lead to premature truncation of BRCA2 as a result of frameshift deletions/insertions or nonsense mutations. Analysis of the mutation distribution along the length of the gene indicates a significant genotype-phenotype correlation. Truncating mutations in families with the highest risk of ovarian cancer relative to breast cancer are clustered in a region of approximately 3.3 kb in exon 11 (P = 0.0004). Published data on mutations in 45 other BRCA2-linked families provide support for this correlation.

Evidence for a familial Wilms' tumour gene (FWT1) on chromosome 17q12-q21.

Wilms' tumour (WT) is one of the most common solid tumours of childhood, occurring in 1 in 10,000 children and accounting for 8% of childhood cancers. It is believed to result from malignant transformation of abnormally persistent renal stem cells (nephrogenic rests) which retain embryonic differentiation potential. Although WT is usually sporadic, approximately one percent occur in families in which susceptibility appears to be inherited as an autosomal dominant trait with incomplete penetrance. Predisposition to other cancers or to the developmental abnormalities associated with sporadic WT is not usually apparent in WT families. The WT1 gene at 11p13 (ref.2), and additional genes on chromosomes 11p15 (ref. 3) and 16q (ref. 4) have been implicated in the development of WT but are not responsible for familial WT. We have carried out a genome linkage search in a large Canadian family with seven confirmed cases of WT. Our results provide strong evidence for the localisation of a familial WT predisposition gene, FWT1, to an 18-centimorgan (cM) interval on chromosome 17q12-q21.

Familial cylindromatosis (turban tumour syndrome) gene localised to chromosome 16q12-q13: evidence for its role as a tumour suppressor gene.

The human skin is a complex organ composed of the surface epidermis, the subjacent dermis (in which blood vessels, lymphatics and nerves are located) and the skin appendages. The latter include hair follicles, sebaceous glands (which secrete lipids that may serve as a permeability barrier, emollient or antimicrobial agent), apocrine glands (which secrete scents) and eccrine glands (which produce sweat for temperature control). Hereditary cylindromatosis (MIM 123850) is a rare autosomal dominant disease characterised by the development of multiple neoplasms originating from the skin appendages. These neoplasms have been termed cylindromas due to their characteristic microscopic architecture and are believed to exhibit apocrine or eccrine differentiation. We have carried out a genome search using two families with this disease, which has provided strong evidence for linkage of cylindromatosis to loci on chromosome 16q12-q13. Using markers close to the cylindromatosis gene, consistent loss of the wild-type allele was observed in 19 tumours from four individuals in the two families, indicating that the gene is likely to be a tumour suppressor gene.

Ovarian cancer risk in BRCA1 carriers is modified by the HRAS1 variable number of tandem repeat (VNTR) locus.

Women who carry a mutation in the BRCA1 gene (on chromosome 17q21), have an 80% risk of breast cancer and a 40% risk of ovarian cancer by the age of 70 (ref. 1). The variable penetrance of BRCA1 suggests that other genetic and non-genetic factors play a role in tumourigenesis in these individuals. The HRAS1 variable number of tandem repeats (VNTR) polymorphism, located 1 kilobase (kb) downstream of the HRAS1 proto-oncogene (chromosome 11p15.5) is one possible genetic modifier of cancer penetrance. Individuals who have rare alleles of the VNTR have an increased risk of certain types of cancers, including breast cancer (2-4). To investigate whether the presence of rare HRAS1 alleles increases susceptibility to hereditary breast and ovarian cancer, we have typed a panel of 307 female BRCA1 carriers at this locus using a PCR-based technique. The risk for ovarian cancer was 2.11 times greater for BRCA1 carriers harbouring one or two rare HRAS1 alleles, compared to carriers with only common alleles (P = 0.015). The magnitude of the relative risk associated with a rare HRAS1 allele was not altered by adjusting for the other known risk factors for hereditary ovarian cancer (5). Susceptibility to breast cancer did not appear to be affected by the presence of rare HRAS1 alleles. This study is the first to show the effect of a modifying gene on the penetrance of an inherited cancer syndrome.

BRCA2 mutations in primary breast and ovarian cancers.

The second hereditary breast cancer gene, BRCA2, was recently isolated. Germline mutations of this gene predispose carriers to breast cancer, and, to a lesser extent, ovarian cancer. Loss of heterozygosity (LOH) at the BRCA2 locus has been observed in 30-40% of sporadic breast and ovarian tumours, implying that BRCA2 may act as a tumour suppressor gene in a proportion of sporadic cases. To define the role of BRCA2 in sporadic breast and ovarian cancer, we screened the entire gene for mutations using a combination of techniques in 70 primary breast carcinomas and in 55 primary epithelial ovarian carcinomas. Our analysis revealed alterations in 2/70 breast tumours and none of the ovarian carcinomas. One alteration found in the breast cancers was a 2-basepair (bp) deletion (4710delAG) which was subsequently shown to be a germline mutation, the other was a somatic missense mutation (Asp3095Glu) of unknown significance. Our results suggest that BRCA2 is a very infrequent target for somatic inactivation in breast and ovarian carcinomas, similar to the results obtained for BRCA1.

Germline mutations of the BRCA1 gene in breast and ovarian cancer families provide evidence for a genotype-phenotype correlation.

Mutations in the BRCA1 gene, discovered in 1994, are associated with an 80-90% lifetime risk of breast cancer. We have analysed 60 families with a history of breast and/or ovarian cancer for germline mutations in BRCA1. Twenty-two different mutations were detected in 32 families (53%), of which 14 are previously unreported. We observed a significant correlation between the location of the mutation in the gene and the ratio of breast to ovarian cancer incidence within each family. Our data suggest a transition in risk such that mutations in the 3' third of the gene are associated with a lower proportion of ovarian cancer. Haplotype analysis supports previous data which suggest some BRCA1 mutation carriers have common ancestors; however, we have found at least two examples where recurrent mutations appear to have arisen independently.

Localization to Xq27 of a susceptibility gene for testicular germ-cell tumours.

Testicular germ-cell tumours (TGCT) affect 1 in 500 men and are the most common cancer in males aged 15-40 in Western European populations. The incidence of TGCT has risen dramatically over the last century. Known risk factors for TGCT include a history of undescended testis (UDT), testicular dysgenesis, infertility, previously diagnosed TGCT (ref. 7) and a family history of the disease. Brothers of men with TGCT have an 8-10-fold risk of developing TGCT (refs 8,9), whereas the relative risk to fathers and sons is fourfold (ref. 9). This familial relative risk is much higher than that for most other types of cancer. We have collected samples from 134 families with two or more cases of TGCT, 87 of which are affected sibpairs. A genome-wide linkage search yielded a heterogeneity lod (hlod) score of 2.01 on chromosome Xq27 using all families compatible with X inheritance. We obtained a hlod score of 4.7 from families with at least one bilateral case, corresponding to a genome-wide significance level of P=0.034. The proportion of families with UDT linked to this locus was 73% compared with 26% of families without UDT (P=0.03). Our results provide evidence for a TGCT susceptibility gene on chromosome Xq27 that may also predispose to UDT.

Constitutional translocation breakpoint mapping by genome-wide paired-end sequencing identifies HACE1 as a putative Wilms tumour susceptibility gene.

BACKGROUND: Localisation of the breakpoints of chromosomal translocations has aided the discovery of several disease genes but has traditionally required laborious investigation of chromosomes by fluorescent in situ hybridisation approaches. Here, a strategy that utilises genome-wide paired-end massively parallel DNA sequencing to rapidly map translocation breakpoints is reported. This method was used to fine map a de novo t(5;6)(q21;q21) translocation in a child with bilateral, young-onset Wilms tumour. METHODS AND RESULTS: Genome-wide paired-end sequencing was performed for approximately 6 million randomly generated approximately 3 kb fragments from constitutional DNA containing the translocation, and six fragments in which one end mapped to chromosome 5 and the other to chromosome 6 were identified. This mapped the translocation breakpoints to within 1.7 kb. Then, PCR assays that amplified across the rearrangement junction were designed to characterise the breakpoints at sequence-level resolution. The 6q21 breakpoint transects and truncates HACE1, an E3 ubiquitin-protein ligase that has been implicated as a somatically inactivated target in Wilms tumourigenesis. To evaluate the contribution of HACE1 to Wilms tumour predisposition, the gene was mutationally screened in 450 individuals with Wilms tumour. One child with unilateral Wilms tumour and a truncating HACE1 mutation was identified. CONCLUSIONS: These data indicate that constitutional disruption of HACE1 likely predisposes to Wilms tumour. However, HACE1 mutations are rare and therefore can only make a small contribution to Wilms tumour incidence. More broadly, this study demonstrates the utility of genome-wide paired-end sequencing in the delineation of apparently balanced chromosomal translocations, for which it is likely to become the method of choice.

LKB1/KRAS mutant lung cancers constitute a genetic subset of NSCLC with increased sensitivity to MAPK and mTOR signalling inhibition.

LKB1/STK11 is a multitasking tumour suppressor kinase. Germline inactivating mutations of the gene are responsible for the Peutz-Jeghers hereditary cancer syndrome. It is also somatically inactivated in approximately 30% of non-small-cell lung cancer (NSCLC). Here, we report that LKB1/KRAS mutant NSCLC cell lines are sensitive to the MEK inhibitor CI-1040 shown by a dose-dependent reduction in proliferation rate, whereas LKB1 and KRAS mutations alone do not confer similar sensitivity. We show that this subset of NSCLC is also sensitised to the mTOR inhibitor rapamycin. Importantly, the data suggest that LKB1/KRAS mutant NSCLCs are a genetically and functionally distinct subset and further suggest that this subset of lung cancers might afford an opportunity for exploitation of anti-MAPK/mTOR-targeted therapies.

Frequent activating FGFR2 mutations in endometrial carcinomas parallel germline mutations associated with craniosynostosis and skeletal dysplasia syndromes.

Endometrial carcinoma is the most common gynecological malignancy in the United States. Although most women present with early disease confined to the uterus, the majority of persistent or recurrent tumors are refractory to current chemotherapies. We have identified a total of 11 different FGFR2 mutations in 3/10 (30%) of endometrial cell lines and 19/187 (10%) of primary uterine tumors. Mutations were seen primarily in tumors of the endometrioid histologic subtype (18/115 cases investigated, 16%). The majority of the somatic mutations identified were identical to germline activating mutations in FGFR2 and FGFR3 that cause Apert Syndrome, Beare-Stevenson Syndrome, hypochondroplasia, achondroplasia and SADDAN syndrome. The two most common somatic mutations identified were S252W (in eight tumors) and N550K (in five samples). Four novel mutations were identified, three of which are also likely to result in receptor gain-of-function. Extensive functional analyses have already been performed on many of these mutations, demonstrating they result in receptor activation through a variety of mechanisms. The discovery of activating FGFR2 mutations in endometrial carcinoma raises the possibility of employing anti-FGFR molecularly targeted therapies in patients with advanced or recurrent endometrial carcinoma.

AutoCSA, an algorithm for high throughput DNA sequence variant detection in cancer genomes.

UNLABELLED: The undertaking of large-scale DNA sequencing screens for somatic variants in human cancers requires accurate and rapid processing of traces for variants. Due to their often aneuploid nature and admixed normal tissue, heterozygous variants found in primary cancers are often subtle and difficult to detect. To address these issues, we have developed a mutation detection algorithm, AutoCSA, specifically optimized for the high throughput screening of cancer samples. AVAILABILITY: http://www.sanger.ac.uk/genetics/CGP/Software/AutoCSA.

Hereditary predisposition to breast cancer.

The breast cancer susceptibility gene on chromosome 17q, BRCA1, has now been isolated. Mutations in this gene have been detected in many families with a predisposition to breast cancer. Most of these mutations result in truncation and presumed inactivation of the BRCA1 protein. A large number of distinct mutations have been reported, although some families have identical mutations, probably due to a founder effect. Certain evidence suggests that mutations positioned towards the 5' end of the gene carry a higher risk of ovarian cancer than those at the 3' end. BRCA1 is infrequently somatically mutated in sporadic breast or ovarian cancer. The BRCA2 gene has been localized to chromosome 13q12-q13. BRCA2 carries a risk of breast cancer similar to that of BRCA1, but is associated with a lower risk of ovarian cancer and a higher risk of male breast cancer. Additional breast cancer susceptibility genes probably exist, but may be difficult to locate by conventional methods.

Prevalence of BRCA1 and BRCA2 gene mutations in patients with early-onset breast cancer.

BACKGROUND: Mutations in the BRCA1 and BRCA2 genes are found in most families with cases of both breast and ovarian cancer or with many cases of early-onset breast cancer. However, in an outbred population, the prevalence of BRCA1 and BRCA2 mutations in patients with breast cancer who were unselected for a family history of this disease has not been determined. METHODS: Mutations in the BRCA1 and BRCA2 genes were detected in blood samples from two population-based series of young patients with breast cancer from Britain. RESULTS: Mutations were detected in 15 (5.9%) of 254 women diagnosed with breast cancer before age 36 years (nine [3.5%] in BRCA1 and six [2.4%] in BRCA2) and in 15 (4.1%) of 363 women diagnosed from ages 36 through 45 years (seven [1.9%] in BRCA1 and eight [2.2%] in BRCA2). Eleven percent (six of 55) of patients with a first-degree relative who developed ovarian cancer or breast cancer by age 60 years were mutation carriers, compared with 45% (five of 11) of patients with two or more affected first- or second-degree relatives. The standardized incidence ratio for breast cancer in mothers and sisters was 365 (five observed and 1.37 expected) for 30 mutation carriers and 199 (64 observed and 32.13 expected) for 587 noncarriers. If we assume recent penetrance estimates, the respective proportions of BRCA1 and BRCA2 mutation carriers are 3.1% and 3.0%, respectively, of patients with breast cancer who are younger than age 50 years, 0.49% and 0.84% of patients with breast cancer who are age 50 years or older, and 0.11% and 0.12% of women in the general population. CONCLUSIONS: Mutations in the BRCA1 and BRCA2 genes make approximately equal contributions to early-onset breast cancer in Britain and account for a small proportion of the familial risk of breast cancer.

Multifactorial analysis of differences between sporadic breast cancers and cancers involving BRCA1 and BRCA2 mutations.

BACKGROUND: We have previously demonstrated that breast cancers associated with inherited BRCA1 and BRCA2 gene mutations differ from each other in their histopathologic appearances and that each of these types differs from breast cancers in patients unselected for family history (i.e., sporadic cancers). We have now conducted a more detailed examination of cytologic and architectural features of these tumors. METHODS: Specimens of tumor tissue (5-microm-thick sections) were examined independently by two pathologists, who were unaware of the case or control subject status, for the presence of cell mitosis, lymphocytic infiltration, continuous pushing margins, and solid sheets of cancer cells; cell nuclei, cell nucleoli, cell necrosis, and cell borders were also evaluated. The resulting data were combined with previously available information on tumor type and tumor grade and further evaluated by multifactorial analysis. All statistical tests are two-sided. RESULTS: Cancers associated with BRCA1 mutations exhibited higher mitotic counts (P = .001), a greater proportion of the tumor with a continuous pushing margin (P<.0001), and more lymphocytic infiltration (P = .002) than sporadic (i.e., control) cancers. Cancers associated with BRCA2 mutations exhibited a higher score for tubule formation (fewer tubules) (P = .0002), a higher proportion of the tumor perimeter with a continuous pushing margin (P<.0001), and a lower mitotic count (P = .003) than control cancers. CONCLUSIONS: Our study has identified key features of the histologic phenotypes of breast cancers in carriers of mutant BRCA1 and BRCA2 genes. This information may improve the classification of breast cancers in individuals with a family history of the disease and may ultimately aid in the clinical management of patients.

Detection of point mutations in N-ras and K-ras genes of human embryonal rhabdomyosarcomas using oligonucleotide probes and the polymerase chain reaction.

Previous studies have demonstrated that genes of the ras family (H, K, and N) can be activated by point mutations at codons 12, 13, and 61. In the present study we have used oligonucleotide probes corresponding to these regions to assess the role of ras gene mutations in the genesis of human rhabdomyosarcoma. To increase the sensitivity of this method the appropriate regions of the three ras genes were first amplified using the polymerase chain reaction. The results show that 35% (5/14) embryonal rhabdomyosarcomas investigated contain mutations in the N-ras or K-ras genes. Thus ras gene mutation is implicated in the development of mesenchymal and embryonal tumors in addition to its previously documented role in epithelial and hematological neoplasia.