Barry Rosen

A conditional knockout resource for the genome-wide study of mouse gene function

Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.

Occult carcinoma in prophylactic oophorectomy specimens: Prevalence and association with BRCA germline mutation status

Prophylactic oophorectomy (PO) is an option for women at increased risk for ovarian carcinoma. In this study the value of intensive pathologic examination of PO specimens and accompanying resected tissues in the identification of occult carcinoma and any association of occult carcinoma with BRCA germline mutation status were ascertained. Specimens from 60 consecutive PO patients, who were not suspected of having any ovarian tumor at the time of surgery, were subjected to standardized, complete pathologic examination in a prospective study over an 8-year period. Extra-ovarian tissues were examined as well, but they were not subject to the same standardized protocol. Any occult carcinoma of the ovaries or fallopian tubes was noted. The BRCA status and follow-up of patients were obtained, if available. Fifty-five of the 60 PO specimens did not show any evidence of malignancy. Of the 32 patients in this group followed for >1 year, all are alive and well. The remaining five patients, all BRCA1 mutation positive, showed occult carcinoma of the ovaries and/or in situ or invasive carcinoma of a fallopian tube. One of these five patients has died of abdominal carcinomatosis; four continue to be well, but follow-up is <4 years in all cases. Occult carcinoma is present in a small proportion of BRCA-positive or unknown PO patients and may be of prognostic significance. The entire ovaries and tubes from PO patients should be submitted for histologic examination to identify malignancy.

BRCA1 and BRCA2 Mutation Analysis of 208 Ashkenazi Jewish Women with Ovarian Cancer

Ovarian cancer is a component of the autosomal-dominant hereditary breast-ovarian cancer syndrome and may be due to a mutation in either the BRCA1 or BRCA2 genes. Two mutations in BRCA1 (185delAG and 5382insC) and one mutation in BRCA2 (6174delT) are common in the Ashkenazi Jewish population. One of these three mutations is present in approximately 2% of the Jewish population. Each mutation is associated with an increased risk of ovarian cancer, and it is expected that a significant proportion of Jewish women with ovarian cancer will carry one of these mutations. To estimate the proportion of ovarian cancers attributable to founding mutations in BRCA1 and BRCA2 in the Jewish population and the familial cancer risks associated with each, we interviewed 213 Jewish women with ovarian cancer at 11 medical centers in North America and Israel and offered these women genetic testing for the three founder mutations. To establish the presence of nonfounder mutations in this population, we also completed the protein-truncation test on exon 11 of BRCA1 and exons 10 and 11 of BRCA2. We obtained a detailed family history on all women we studied who had cancer and on a control population of 386 Ashkenazi Jewish women without ovarian or breast cancer. A founder mutation was present in 41.3% of the women we studied. The cumulative incidence of ovarian cancer to age 75 years was found to be 6.3% for female first-degree relatives of the patients with ovarian cancer, compared with 2.0% for the female relatives of healthy controls (relative risk 3.2; 95% CI 1.5-6.8; P=.002). The relative risk to age 75 years for breast cancer among the female first-degree relatives was 2.0 (95% CI 1.4-3.0; P=.0001). Only one nonfounder mutation was identified (in this instance, in a woman of mixed ancestry), and the three founding mutations accounted for most of the observed excess risk of ovarian and breast cancer in relatives.

Impact of Oophorectomy on Cancer Incidence and Mortality in Women With a BRCA1 or BRCA2 Mutation

PURPOSE The purposes of this study were to estimate the reduction in risk of ovarian, fallopian tube, or peritoneal cancer in women with a BRCA1 or BRCA2 mutation after oophorectomy, by age of oophorectomy; to estimate the impact of prophylactic oophorectomy on all-cause mortality; and to estimate 5-year survival associated with clinically detected ovarian, occult, and peritoneal cancers diagnosed in the cohort. PATIENTS AND METHODS Women with a BRCA1 or BRCA2 mutation were identified from an international registry; 5,783 women completed a baseline questionnaire and ≥ one follow-up questionnaires. Women were observed until either diagnosis of ovarian, fallopian tube, or peritoneal cancer, death, or date of most recent follow-up. Hazard ratios (HRs) for cancer incidence and all-cause mortality associated with oophorectomy were evaluated using time-dependent survival analyses. RESULTS After an average follow-up period of 5.6 years, 186 women developed either ovarian (n = 132), fallopian (n = 22), or peritoneal (n = 32) cancer, of whom 68 have died. HR for ovarian, fallopian, or peritoneal cancer associated with bilateral oophorectomy was 0.20 (95% CI, 0.13 to 0.30; P < .001). Among women who had no history of cancer at baseline, HR for all-cause mortality to age 70 years associated with an oophorectomy was 0.23 (95% CI, 0.13 to 0.39; P < .001). CONCLUSION Preventive oophorectomy was associated with an 80% reduction in the risk of ovarian, fallopian tube, or peritoneal cancer in BRCA1 or BRCA2 carriers and a 77% reduction in all-cause mortality.

International variation in rates of uptake of preventive options in BRCA1 and BRCA2 mutation carriers

Several options for cancer prevention are available for women with a BRCA1 or BRCA2 mutation, including prophylactic surgery, chemoprevention and screening. The authors report on preventive practices in women with mutations from 9 countries and examine differences in uptake according to country. Women with a BRCA1 or BRCA2 mutation were contacted after receiving their genetic test result and were questioned regarding their preventive practices. Information was recorded on prophylactic mastectomy, prophylactic oophorectomy, use of tamoxifen and screening (MRI and mammography). Two thousand six hundred seventy‐seven women with a BRCA1 or BRCA2 mutation from 9 countries were included. The follow‐up questionnaire was completed a mean of 3.9 years (range 1.5–10.3 years) after genetic testing. One thousand five hundred thirty‐one women (57.2%) had a bilateral prophylactic oophorectomy. Of the 1,383 women without breast cancer, 248 (18.0%) had had a prophylactic bilateral mastectomy. Among those who did not have a prophylactic mastectomy, only 76 women (5.5%) took tamoxifen and 40 women (2.9%) took raloxifene for breast cancer prevention. Approximately one‐half of the women at risk for breast cancer had taken no preventive option, relying solely on screening. There were large differences in the uptake of the different preventive options by country of residence. Prophylactic oophorectomy is now generally accepted by women and their physicians as a cancer preventive measure. However, only the minority of women with a BRCA1 or BRCA2 mutation opt for prophylactic mastectomy or take tamoxifen for the prevention of hereditary breast cancer. Approximately one‐half of women at risk for breast cancer rely on screening alone.

Tubal ligation and risk of ovarian cancer in carriers of BRCA1 or BRCA2 mutations: a case-control study

BACKGROUND In several case-control and prospective studies, tubal ligation has been associated with a decreased risk of invasive epithelial ovarian cancer. We aimed to assess the potential of tubal ligation in reducing the risk of ovarian cancer in women who carry predisposing mutations in the BRCA1 or BRCA2 genes. METHODS We did a matched case-control study among women from Canada, the USA, and the UK who had undergone genetic testing and who carried a pathogenic mutation in BRCA1 or BRCA2. Cases were 232 women with a history of invasive ovarian cancer, and controls were 232 women without ovarian cancer, and who had both ovaries intact. Cases and controls were matched for year of birth, country of residence, and mutation (BRCA1 or BRCA2). The odds ratio for developing ovarian cancer was estimated for tubal ligation, adjusting for oral contraceptive use, parity, history of breast cancer, and ethnic group. FINDINGS In an unadjusted analysis among BRCA1 carriers, significantly fewer cases than controls had ever had tubal ligation (30 of 173 [18%] vs 60 of 173 [35%], odds ratio 0.37 [95% CI 0.21-0.63]; p=0.0003). After adjustment for oral contraceptive use, parity, history of breast cancer and ethnic group, the odds ratio was 0.39 (p=0.002). Combination of tubal ligation and past use of an oral contraceptive was associated with an odds ratio of 0.28 (0.15-0.52). No protective effect of tubal ligation was seen among carriers of the BRCA2 mutation. INTERPRETATION Tubal ligation is a feasible option to reduce the risk of ovarian cancer in women with BRCA1 mutations who have completed childbearing.

Frequencies of BRCA1 and BRCA2 mutations among 1,342 unselected patients with invasive ovarian cancer.

BACKGROUND The heritable fraction of ovarian cancer exceeds that of any other common adult cancer. Most inherited cases of ovarian cancer are due to a germline mutation in BRCA1 or BRCA2. It is important to have an accurate estimate of the proportion of ovarian cancer patients who carry a mutation and the specific factors which predict the presence of a mutation. METHODS We tested a population-based series of 1342 unselected patients diagnosed with invasive ovarian cancer between 1995-1999 and 2002-2004 in Ontario, Canada, for germline mutations in BRCA1 and BRCA2. The two genes were tested in their entirety, using a range of techniques, including multiplex ligation-dependent probe amplification (MLPA). RESULTS Among the 1342 women, 176 women carried a mutation (107 in BRCA1, 67 in BRCA2, and two in both genes) for a combined mutation frequency of 13.3%. Seven deletions were identified using MLPA (3.9% of all detected mutations). The prevalence of mutations was particularly high among women diagnosed in their forties (24.0%), in women with serous ovarian cancer (18.0%) and women of Italian (43.5%), Jewish (30.0%) or Indo-Pakistani origin (29.4%). A mutation was seen in 33.9% of women with a first-degree relative with breast or ovarian cancer and in 7.9% of women with no first-degree relative with breast or ovarian cancer. No mutation was seen in women with mucinous carcinoma. CONCLUSIONS BRCA1 and BRCA2 mutations are common in women with invasive ovarian cancer. All women diagnosed with invasive non-mucinous ovarian cancer should be considered to be candidates for genetic testing.

The European dimension for the mouse genome mutagenesis program

The European Mouse Mutagenesis Consortium is the European initiative contributing to the international effort on functional annotation of the mouse genome. Its objectives are to establish and integrate mutagenesis platforms, gene expression resources, phenotyping units, storage and distribution centers and bioinformatics resources. The combined efforts will accelerate our understanding of gene function and of human health and disease.

Reproductive risk factors for ovarian cancer in carriers of BRCA1 or BRCA2 mutations: a case-control study

BACKGROUND Several of the known risk factors for ovarian cancer are thought to act through their effects on ovulation and the menstrual cycle, such as parity, breastfeeding, and use of oral contraceptives. We aimed to assess the effect of these three risk factors, and of tubal ligation, on the risk of ovarian cancer in women who carry a mutation in the BRCA1 or BRCA2 genes. METHODS We did a matched case-control study in women who were found to carry a pathogenetic mutation in BRCA1 or BRCA2. Participants were derived from a population-based study of ovarian cancer in Ontario, Canada, and from an international registry of mutation carriers based in Toronto, ON, Canada. All participants completed a written questionnaire that detailed their reproductive history. Women with invasive ovarian cancer and controls were matched on year of birth, country of residence, mutation (BRCA1 or BRCA2), and history of breast cancer. The odds ratios and 95% CI for ovarian cancer were estimated with respect to use of oral contraceptives, parity, breastfeeding, and tubal ligation. FINDINGS Questionnaires were completed by 799 women with a history of invasive ovarian cancer (670 with BRCA1 mutations, 128 with BRCA2 mutations, and one with a mutation in both genes), and controls were 2424 women without ovarian cancer (2043 with BRCA1 mutations, 380 with BRCA2 mutations, and one with a mutation in both genes). Use of oral contraceptives reduced the risk of ovarian cancer in carriers of BRCA1 mutations (odds ratio 0.56 [95% CI 0.45-0.71]; p<0.0001) and carriers of BRCA2 mutations (0.39 [0.23-0.66]; p=0.0004). Parity was associated with a reduced risk for carriers of BRCA1 mutations (0.67 [0.46-0.96]; p=0.03), but with an increased risk for those with BRCA2 mutations (2.74 [1.18-6.41]; p=0.02). Breastfeeding was associated with a reduced risk for carriers of BRCA1 mutations (0.74 [0.56-0.97]; p=0.03). An effect of similar magnitude was seen for carriers of BRCA2 mutations (0.72 [0.41-1.29]; p=0.27), but this was not statistically significant. The association with tubal ligation was not significant for carriers of BRCA1 mutations (0.80 [0.59-1.08]; p=0.15), or for carriers of BRCA2 mutations (0.63 [0.34-1.15]; p=0.13). INTERPRETATION Oral contraceptives could be used as a means to prevent ovarian cancer in carriers of BRCA1 and BRCA2 mutations. The possible adverse effect of parity on ovarian-cancer risk in women with a BRCA2 mutation needs further study.

The fallopian tube: primary site of most pelvic high-grade serous carcinomas.

Epithelial ovarian cancer is the most common cause of mortality from gynecologic malignancy, and most of epithelial cancers are of serous type. The site of origin of pelvic high-grade serous carcinoma has been the subject of debate for 60 years. This paper reviews the evidence that pelvic serous carcinoma originates from the fallopian tube mucosa and puts forward a theory that inflammation in the tube, caused by menstrual cytokines or infection, is critical to the genesis of these tumors. Other risk factors for pelvic serous carcinoma will be reviewed, including oral contraceptive use, parity, infertility, and tubal ligation. Studies were identified for this review by searching the English language literature in the MEDLINE database between the years 1995 and 2007 using the following keywords: fallopian tube neoplasia, ovarian serous adenocarcinoma, pregnancy, oral contraceptive, infertility, pelvic inflammatory disease, cytokines, menstruation, and tubal ligation, followed by an extensive review of bibliographies from articles found through the search. The clinical implications of this theory are discussed, and a change in surgical practice is recommended, with salpingectomy at the time of simple hysterectomy. This theory also has implications for the development of new methods of screening for pelvic serous carcinomas, as there are no screening methods that are currently available to find this form of cancer in an early stage. Inflammatory markers could be detected in the vagina from the fallopian tube indicating possible chronic inflammation and a risk factor for mutagenesis leading to serous carcinoma.