Performance of high-throughput DNA quantification methods

BACKGROUND: The accuracy and precision of estimates of DNA concentration are critical factors for efficient use of DNA samples in high-throughput genotype and sequence analyses. We evaluated the performance of spectrophotometric (OD) DNA quantification, and compared it to two fluorometric quantification methods, the PicoGreen(® )assay (PG), and a novel real-time quantitative genomic PCR assay (QG) specific to a region at the human BRCA1 locus. Twenty-Two lymphoblastoid cell line DNA samples with an initial concentration of ~350 ng/uL were diluted to 20 ng/uL. DNA concentration was estimated by OD and further diluted to 5 ng/uL. The concentrations of multiple aliquots of the final dilution were measured by the OD, QG and PG methods. The effects of manual and robotic laboratory sample handling procedures on the estimates of DNA concentration were assessed using variance components analyses. RESULTS: The OD method was the DNA quantification method most concordant with the reference sample among the three methods evaluated. A large fraction of the total variance for all three methods (36.0–95.7%) was explained by sample-to-sample variation, whereas the amount of variance attributable to sample handling was small (0.8–17.5%). Residual error (3.2–59.4%), corresponding to un-modelled factors, contributed a greater extent to the total variation than the sample handling procedures. CONCLUSION: The application of a specific DNA quantification method to a particular molecular genetic laboratory protocol must take into account the accuracy and precision of the specific method, as well as the requirements of the experimental workflow with respect to sample volumes and throughput. While OD was the most concordant and precise DNA quantification method in this study, the information provided by the quantitative PCR assay regarding the suitability of DNA samples for PCR may be an essential factor for some protocols, despite the decreased concordance and precision of this method

A common founder for the V126D CDKN2A mutation in seven North American melanoma-prone families

One of the most common melanoma-related CDKN2A mutations reported in North America is the V126D mutation. We examined nine markers surrounding CDKN2A in three American and four Canadian families carrying the V126D mutation. All seven families had a haplotype consistent with a common ancestor/founder for this mutation. In addition, the mutation appears to have originated 34–52 generations ago (1-LOD-unit support interval 13–98 generations). © 2001 Cancer Research Campaign http:///www.bjcancer.co

Prevalence of BRCA1 and BRCA2 Jewish mutations in Spanish breast cancer patients

We screened the 185delAG and 5382insC (BRCA1) and the 6174delT (BRCA2) mutation in 298 Spanish women with breast cancer. Two women (one with Sephardic ancestors) presented the 185delAG mutation and the same haplotype reported in Ashkenazim with this mutation. This suggests a common origin of the 185delAG in both Sephardic and Ashkenazi populations. © 1999 Cancer Research Campaig

Founder populations and their uses for breast cancer genetics

Numerous founder mutations have been reported in BRCA1 and BRCA2. For genetic screening of a population with a founder mutation, testing can be targeted to the mutation, allowing for a more rapid and less expensive test. In addition, more precise estimates of the prior probability of carrying a mutation and of the likelihood of a mutation carrier developing cancer should be possible. For a given founder mutation a large number of carriers are available, so that focused scientific studies of penetrance, expression, and genetic and environmental modifiers of risk can be performed. Finally, founder populations may be a powerful resource to localize additional breast cancer susceptibility loci, because of the reduction in locus heterogeneity

Estimating the survival benefits gained from providing national cancer genetic services to women with a family history of breast cancer

The aim of this paper is to compare a service offering genetic testing and presymptomatic surveillance to women at increased risk of developing breast cancer with its predecessor of no service at all in terms of survival and quality-adjusted survival (QALYs) by means of a Markov cohort chain simulation model. Genetic assessment and presymptomatic care provided between 0.07-1.61 mean additional life years and 0.05-1.67 mean QALYs over no services. Prophylactic surgery and surveillance extended mean life expectancy by 0.41-1.61 and 0.32-0.99 years, respectively over no services for high-risk women. Model outcomes were sensitive to all the parameters varied in the sensitivity analysis. Providing cancer genetic services increase survival and as long as services do not induce adverse psychological effects they also provide more QALYs. The greatest survival and QALY benefits were found for women with identified mutations. As more cancer genes are identified, the survival and cost-effectiveness of genetic services will improve. Although mastectomy provided most additional life years, when quality of life was accounted for oophorectomy was the optimal strategy. Delayed entry into coordinated genetic services was found to diminish the average survival and QALY gains for a woman utilising these services

Oral contraceptive use and ovarian cancer risk among carriers of BRCA1 or BRCA2 mutations

Women with mutations of the genes BRCA1 or BRCA2 are at increased risk of ovarian cancer. Oral contraceptives protect against ovarian cancer in general, but it is not known whether they protect against the disease in carriers of these mutations. We obtained self-reported lifetime histories of oral contraceptive use from 451 women who carried mutations of BRCA1 or BRCA2. We used conditional logistic regression to estimate the odds ratios associated with oral contraceptive use, comparing the histories of 147 women with ovarian cancer (cases) to those of 304 women without ovarian cancer (controls) who were matched to cases on year of birth, country of residence and gene (BRCA1 vs BRCA2). Reference ages for controls had to exceed the ages at diagnosis of their matched cases. After adjusting for parity, the odds-ratio for ovarian cancer associated with use of oral contraceptives for at least 1 year was 0.85 (95 percent confidence interval, 0.53-1.36). The risk decreased by 5% (1-9%) with each year of use (P for trend=0.01). Use for 6 or more years was associated with an odds-ratio of 0.62 (0.35-1.09). These data support the hypothesis that long-term oral contraceptive use reduces the risk of ovarian cancer among women who carry mutations of BRCA1 or BRCA2

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-µm-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. [J Natl Cancer Inst 1998;90:1138-45

Randomised trial of a decision aid and its timing for women being tested for a BRCA1/2 mutation

Contains fulltext : 57882.pdf (publisher's version ) (Closed access)The aim of the study was to evaluate the impact of a decision aid (DA) and its timing in women being tested for a BRCA1/2 mutation. Women with and without a previous history of cancer were included after blood sampling for genetic testing. The DA consisted of a brochure and video providing information on screening and prophylactic surgery. To evaluate the impact of the DA, women were randomised to the DA group (n=184), receiving the DA 2 weeks after blood sampling, or to the control group (n=184). To evaluate the impact of timing, mutation carriers who had received the DA before the test result (n=47) were compared to mutation carriers who received the DA after the test result (n=42). Data were collected on well-being, treatment choice, decision and information related outcomes. The impact of the DA was measured 4 weeks after blood sampling. The impact of timing was measured 2 weeks after a positive test result. The DA had no impact on well-being. Regarding decision related outcomes, the DA group more frequently considered prophylactic surgery (P=0.02) corroborated with higher valuations (P=0.04). No differences were found for the other decision related outcomes. Regarding information related outcomes, the DA group felt better informed (P=0.00), was more satisfied with the information (P=0.00), and showed more accurate risk perceptions. Timing of the DA had no effect on any of the outcomes. No interactions were found between the DA and history of cancer. In conclusion, women being tested for a BRCA1/2 mutation benefit from the DA on information related outcomes. Because timing had no effect, the DA is considered useful either before or after the test result

Log odds of carrying an Ancestral Mutation in BRCA1 or BRCA2 for a Defined personal and family history in an Ashkenazi Jewish woman (LAMBDA)

INTRODUCTION: Ancestral mutations in BRCA1 and BRCA2 are common in people of Ashkenazi Jewish descent and are associated with a substantially increased risk of breast and ovarian cancer. Women considering mutation testing usually have several personal and family cancer characteristics, so predicting mutation status from one factor alone could be misleading. The aim of this study was to develop a simple algorithm to estimate the probability that an Ashkenazi Jewish woman carries an ancestral mutation, based on multiple predictive factors. METHODS: We studied Ashkenazi Jewish women with a personal or family history of breast or ovarian cancer and living in Melbourne or Sydney, Australia, or with a previous diagnosis of breast or ovarian cancer and living in the UK. DNA samples were tested for the germline mutations 185delAG and 5382insC in BRCA1, and 6174delT in BRCA2. Logistic regression was used to identify, and to estimate the predictive strength of, major determinants. RESULTS: A mutation was detected in 64 of 424 women. An algorithm was developed by combining our findings with those from similar analyses of a large study of unaffected Jewish women in Washington. Starting with a baseline score, a multiple of 0.5 (based on the logistic regression estimates) is added for each predictive feature. The sum is the estimated log odds ratio that a woman is a carrier, and is converted to a probability by using a table. There was good internal consistency. CONCLUSIONS: This simple algorithm might be useful in the clinical and genetic counselling setting. Comparison and validation in other settings should be sought