Sequencing-based functional assays for classification of BRCA2 variants in mouse ESCs.

Sequencing of genes, such as BRCA1 and BRCA2, is recommended for individuals with a personal or family history of early onset and/or bilateral breast and/or ovarian cancer or a history of male breast cancer. Such sequencing efforts have resulted in the identification of more than 17,000 BRCA2 variants. The functional significance of most variants remains unknown; consequently, they are called variants of uncertain clinical significance (VUSs). We have previously developed mouse embryonic stem cell (mESC)-based assays for functional classification of BRCA2 variants. We now developed a next-generation sequencing (NGS)-based approach for functional evaluation of BRCA2 variants using pools of mESCs expressing 10-25 BRCA2 variants from a given exon. We use this approach for functional evaluation of 223 variants listed in ClinVar. Our functional classification of BRCA2 variants is concordant with the classification reported in ClinVar or those reported by other orthogonal assays.

A computational model for classification of BRCA2 variants using mouse embryonic stem cell-based functional assays.

Sequencing-based genetic tests to identify individuals at increased risk of hereditary breast and ovarian cancers have resulted in the identification of more than 40,000 sequence variants of BRCA1 and BRCA2. A majority of these variants are considered to be variants of uncertain significance (VUS) because their impact on disease risk remains unknown, largely due to lack of sufficient familial linkage and epidemiological data. Several assays have been developed to examine the effect of VUS on protein function, which can be used to assess their impact on cancer susceptibility. In this study, we report the functional characterization of 88 BRCA2 variants, including several previously uncharacterized variants, using a well-established mouse embryonic stem cell (mESC)-based assay. We have examined their ability to rescue the lethality of Brca2 null mESC as well as sensitivity to six DNA damaging agents including ionizing radiation and a PARP inhibitor. We have also examined the impact of BRCA2 variants on splicing. In addition, we have developed a computational model to determine the probability of impact on function of the variants that can be used for risk assessment. In contrast to the previous VarCall models that are based on a single functional assay, we have developed a new platform to analyze the data from multiple functional assays separately and in combination. We have validated our VarCall models using 12 known pathogenic and 10 neutral variants and demonstrated their usefulness in determining the pathogenicity of BRCA2 variants that are listed as VUS or as variants with conflicting functional interpretation.

Calibration of Pathogenicity Due to Variant-Induced Leaky Splicing Defects by Using BRCA2 Exon 3 as a Model System.

BRCA2 is a clinically actionable gene implicated in breast and ovarian cancer predisposition that has become a high priority target for improving the classification of variants of unknown significance (VUS). Among all BRCA2 VUS, those causing partial/leaky splicing defects are the most challenging to classify because the minimal level of full-length (FL) transcripts required for normal function remains to be established. Here, we explored BRCA2 exon 3 (BRCA2e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for BRCA2 haploinsufficiency. In silico predictions, minigene splicing assays, patients' RNA analyses, a mouse embryonic stem cell (mESC) complementation assay and retrieval of patient-related information were combined to determine the minimal requirement of FL BRCA2 transcripts. Of 100 BRCA2e3 variants tested in the minigene assay, 64 were found to be spliceogenic, causing mild to severe RNA defects. Splicing defects were also confirmed in patients' RNA when available. Analysis of a neutral leaky variant (c.231T>G) showed that a reduction of approximately 60% of FL BRCA2 transcripts from a mutant allele does not cause any increase in cancer risk. Moreover, data obtained from mESCs suggest that variants causing a decline in FL BRCA2 with approximately 30% of wild-type are not pathogenic, given that mESCs are fully viable and resistant to DNA-damaging agents in those conditions. In contrast, mESCs producing lower relative amounts of FL BRCA2 exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of BRCA2 variants affecting the splicing pattern of other essential exons. SIGNIFICANCE: These findings demonstrate that BRCA2 tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of BRCA2 leaky splicing variants, some of which may not increase cancer risk.

Functional evaluation of five BRCA2 unclassified variants identified in a Sri Lankan cohort with inherited cancer syndromes using a mouse embryonic stem cell-based assay.

Next-generation sequencing of Sri Lankan families with inherited cancer syndromes resulted in the identification of five BRCA2 variants of unknown clinical significance. Interpreting such variants poses significant challenges for both clinicians and patients. Using a mouse embryonic stem cell-based functional assay, we found I785V, N830D, and K2077N to be functionally indistinguishable from wild-type BRCA2. Specific but mild sensitivity to olaparib and reduction in homologous recombination (HR) efficiency suggest partial loss of function of the A262T variant. This variant is located in the N-terminal DNA binding domain of BRCA2 that can facilitate HR by binding to dsDNA/ssDNA junctions. P3039P is clearly pathogenic because of premature protein truncation caused by exon 23 skipping. These findings highlight the value of mouse embryonic stem cell-based assays for determining the functional significance of variants of unknown clinical significance and provide valuable information regarding risk estimation and genetic counseling of families carrying these BRCA2 variants.

Synthetic viability by BRCA2 and PARP1/ARTD1 deficiencies.

Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib has been approved for treatment of advanced ovarian cancer associated with BRCA1 and BRCA2 mutations. BRCA1- and BRCA2-mutated cells, which are homologous recombination (HR) deficient, are hypersensitive to PARPi through the mechanism of synthetic lethality. Here we examine the effect of PARPi on HR-proficient cells. Olaparib pretreatment, PARP1 knockdown or Parp1 heterozygosity of Brca2(cko/ko) mouse embryonic stem cells (mESCs), carrying a null (ko) and a conditional (cko) allele of Brca2, results in viable Brca2(ko/ko) cells. PARP1 deficiency does not restore HR in Brca2(ko/ko) cells, but protects stalled replication forks from MRE11-mediated degradation through its impaired recruitment. The functional consequence of Parp1 heterozygosity on BRCA2 loss is demonstrated by a significant increase in tumorigenesis in Brca2(cko/cko) mice. Thus, while olaparib efficiently kills BRCA2-deficient cells, we demonstrate that it can also contribute to the synthetic viability if PARP is inhibited before BRCA2 loss.

Updated US Department of Agriculture Food Patterns meet goals of the 2010 dietary guidelines.

The US Department of Agriculture Food Patterns were updated for the 2010 Dietary Guidelines for Americans to meet new nutrition goals and incorporate results of food pattern modeling requested by the Dietary Guidelines Advisory Committee. The purpose of this article is to describe the process used and changes in the updated patterns. Changes include renaming the Meat and Beans and Milk Groups to the Protein Foods and Dairy Groups, respectively, to be more encompassing of foods in each. Vegetable subgroups now provide more achievable intake recommendations. Calcium-fortified soymilk is now included in the Dairy Group because of its similarity to foods in that group. Increased amounts of seafoods are recommended in the Protein Foods Group, balanced by decreased amounts of meat and poultry. A limit on calories from solid fats and added sugars is included, replacing the previous discretionary calorie allowance and emphasizing the need to choose nutrient-dense forms of foods. Lacto-ovo vegetarian and vegan patterns that meet nutrition goals were created by making substitutions in the Protein Foods Group, and for vegan patterns, in the Dairy Group. Patterns identify food choices that meet nutritional needs within energy allowances and encourage choosing a variety of foods. They rely on foods in nutrient-dense forms, including a limited amount of calories from solid fats and added sugars. The Food Patterns provide a useful template for educating consumers about healthful food choices while highlighting a large gap between choices many Americans make and healthy eating patterns.

Impact of typical rather than nutrient-dense food choices in the US Department of Agriculture Food Patterns.

BACKGROUND: The US Department of Agriculture (USDA) Food Patterns, released as part of the 2010 Dietary Guidelines for Americans, are designed to meet nutrient needs without exceeding energy requirements. They identify amounts to consume from each food group and recommend that nutrient-dense forms-lean or low-fat, without added sugars or salt-be consumed. Americans fall short of most food group intake targets and do not consume foods in nutrient-dense forms. Intake of calories from solid fats and added sugars exceed maximum limits by large margins. OBJECTIVE: Our aim was to determine the potential effect on meeting USDA Food Pattern nutrient adequacy and moderation goals if Americans consumed the recommended quantities from each food group, but did not implement the advice to select nutrient-dense forms of food and instead made more typical food choices. DESIGN: Food-pattern modeling analysis using the USDA Food Patterns, which are structured to allow modifications in one or more aspects of the patterns, was used. Nutrient profiles for each food group were modified by replacing each nutrient-dense representative food with a similar but typical choice. Typical nutrient profiles were used to determine the energy and nutrient content of the food patterns. RESULTS: Moderation goals are not met when amounts of food in the USDA Food Patterns are followed and typical rather than nutrient-dense food choices are made. Energy, total fat, saturated fat, and sodium exceed limits in all patterns, often by substantial margins. With typical choices, calories were 15% to 30% (ie, 350 to 450 kcal) above the target calorie level for each pattern. Adequacy goals were not substantially affected by the use of typical food choices. CONCLUSIONS: If consumers consume the recommended quantities from each food group and subgroup, but fail to choose foods in low-fat, no-added-sugars, and low-sodium forms, they will not meet the USDA Food Patterns moderation goals or the 2010 Dietary Guidelines for Americans.

The US Department of Agriculture Automated Multiple-Pass Method reduces bias in the collection of energy intakes.

BACKGROUND: The US Department of Agriculture Automated Multiple-Pass Method (AMPM) is used for collecting 24-h dietary recalls in What We Eat In America, the dietary interview component of the National Health and Nutrition Examination Survey. Because the data have important program and policy applications, it is essential that the validity of the method be tested. OBJECTIVE: The accuracy of the AMPM was evaluated by comparing reported energy intake (EI) with total energy expenditure (TEE) by using the doubly labeled water (DLW) technique. DESIGN: The 524 volunteers, aged 30-69 y, included an equal number of men and women recruited from the Washington, DC, area. Each subject was dosed with DLW on the first day of the 2-wk study period; three 24-h recalls were collected during the 2-wk period by using the AMPM. The first recall was conducted in person, and subsequent recalls were over the telephone. RESULTS: Overall, the subjects underreported EI by 11% compared with TEE. Normal-weight subjects [body mass index (in kg/m(2)) < 25] underreported EI by <3%. By using a linear mixed model, 95% CIs were determined for the ratio of EI to TEE. Approximately 78% of men and 74% of women were classified as acceptable energy reporters (within 95% CI of EI:TEE). Both the percentage by which energy was underreported and the percentage of subjects classified as low energy reporters (<95% CI of EI:TEE) were highest for subjects classified as obese (body mass index > 30). CONCLUSIONS: Although the AMPM accurately reported EIs in normal-weight subjects, research is warranted to enhance its accuracy in overweight and obese persons.

Effect of snacking frequency on adolescents' dietary intakes and meeting national recommendations.

PURPOSE: To determine how snacking level impacts intake of nutrients and food groups and assists in meeting recommendations outlined in the U.S. Department of Agriculture's MyPyramid Food Guidance System. METHODS: Dietary data based on 24-hour recall from 4357 adolescents 12-19 years of age participating in the National Health and Nutrition Examination Survey 2001-2004 (NHANES) were analyzed. Regression analyses were applied to examine the effect of snacking on nutrient and food group intake and to determine its effect on the likelihood of meeting MyPyramid recommendations. RESULTS: Food energy, carbohydrate, total sugars, and vitamin C intake were positively associated, whereas protein and fat intake were negatively associated, with snacking frequency. Fruit intake increased, whereas solid fat intake decreased, as snacking incidence rose. Increasing snacking frequency was also associated with a greater likelihood of meeting milk and oil recommendations for boys and meeting fruit recommendations for both genders. Non-Hispanic black adolescents were less likely to meet their milk recommendations at low and high snacking levels and more likely to meet their fruit recommendations at high levels only. Foods consumed as snacks provided 12-39% of the day's total number of portions of the five MyPyramid food groups, 35% of total discretionary calorie intake, and 43% of total added sugar intake. CONCLUSIONS: Snacking frequency affects intake of macronutrients and a few micronutrients and promotes consumption of fruits. Top food choices for snacks provide an excess of discretionary calories in the form of added sugars and fats. Modification of these choices would assist adolescents in consuming diets more consistent with national recommendations.

Older adults who use vitamin/mineral supplements differ from nonusers in nutrient intake adequacy and dietary attitudes.

OBJECTIVE: To measure nutrient intake adequacy of vitamin/mineral supplement users and nonusers aged 51 years and older, determine the efficacy of supplement practices in compensating for dietary deficits, and identify predictors of supplement use. DESIGN: Analyses of two 24-hour recalls, demographic variables, and attitude questions collected during the Continuing Survey of Food Intakes by Individuals and Diet and Health Knowledge Survey in 1994 to 1996. Data were weighted to be representative of older Americans. SUBJECTS: Four thousand three hundred eighty-four adults aged 51 years and older (1,777 daily supplement users, 428 infrequent users, and 2,179 nonusers) residing in households in the United States. STATISTICAL ANALYSES: Usual nutrient intake distributions were estimated using the Iowa State University method. The Estimated Average Requirement (EAR) cutpoint method was applied to determine the proportion of older adults not meeting requirements before and after accounting for nutrient intake from supplements. Student t tests were used to assess differences between users and nonusers. Logistic regression was used to determine sociodemographic and attitudinal predictors of supplement use. RESULTS: For one or more of the sex-age groups studied, a significantly smaller proportion of supplement users than nonusers had intakes from food alone below the EAR for vitamins A, B-6, and C; folate; zinc; and magnesium. Even so, less than 50% of both users and nonusers met the EAR for folate, vitamin E, and magnesium from food sources alone. Overall, supplements improved the nutrient intake of older adults. After accounting for the contribution of supplements, 80% or more of users met the EAR for vitamins A, B-6, B-12, C, and E; folate; iron; and zinc, but not magnesium. However, some supplement users, particularly men, exceeded Tolerable Upper Intake Levels for iron and zinc and a small percentage of women exceeded the Tolerable Upper Intake Level for vitamin A. Significant sociodemographic factors related to supplement use for older men were age group, metropolitan area, and educational status. Race, region, smoking status, and vegetarian status were significant factors for women. Attitude about the importance of following a healthful diet was a consistent predictor of supplement use for both men and women. CONCLUSIONS: A large proportion of older adults do not consume sufficient amounts of many nutrients from foods alone. Supplements compensate to some extent, but only an estimated half of this population uses them daily. These widespread inadequacies should be considered when developing recommendations for supplement use for clients in this age group. Modifying dietary attitudes may result in a higher rate of supplement use in this at-risk population.

Tumor resection cavity administered iodine-131-labeled antitenascin 81C6 radioimmunotherapy in patients with malignant glioma: neuropathology aspects.

INTRODUCTION: The neurohistological findings in patients treated with targeted beta emitters such as (131)I are poorly described. We report a histopathologic analysis from patients treated with combined external beam therapy and a brachytherapy consisting of a (131)I-labeled monoclonal antibody (mAb) injected into surgically created resection cavities during brain tumor resections. METHODS: Directed tissue samples of the cavity walls were obtained because of suspected tumor recurrence from 28 patients. Samples and clinical follow-up were evaluated on all patients (Group A) based on total radiation dose received and a subset of these (n=18; Group B, proximal therapy subset) who had received external beam therapy within

A new portion size estimation aid for wedge-shaped foods.

Dietary surveys are important for understanding food consumption patterns in national studies, clinical research, and patient counseling. Portion size estimation aids usually increase accuracy of consumers' reports of the amount of food they ate in dietary surveys. However, wedge-shaped foods (eg, pie, cake, and pizza) pose special problems; this shape is not easily estimated by previously available portion size estimation aids. In focus groups, respondents indicate they need a portion size estimation aid that is easier to use than a ruler for portion estimation of wedge-shaped foods. An adjustable wedge was developed and tested with 320 respondents, ages 18 to 65 years, of various races and education levels, and both sexes, in four states using multiple sizes and types of wedge-shaped foods. The accuracy of portion size estimation with the two portion size estimation aids was assessed. The adjustable wedge gave similar results to the ruler for most portions tested, but was more accurate (P<0.05) than the ruler in approximately one third of comparisons. Both aids resulted in substantial misestimation, indicating that regardless of aid some people have difficulty estimating portions of wedge-shaped foods. The adjustable wedge was easy for people to use and can be recommended as an option for estimating portion size of wedge-shaped foods.

Accuracy of reporting dietary intake using various portion-size aids in-person and via telephone.

OBJECTIVE: This study compared the accuracy with which respondents reported food intake using different portion-size estimation aids (PSEAs) and determined whether the interview technique affected the accuracy of reporting. DESIGN: Participants chose preweighed foods from a buffet line, leftovers were weighed, and actual amounts eaten were calculated. The next day during dietary interviews, participants estimated amounts eaten using one of four procedures, including telephone or in-person interviews, two-dimensional or three-dimensional PSEAs, and guiding or not guiding participants to certain aids. SUBJECTS: A total of 120 persons, ages 18 to 65 years, representing both genders were recruited through advertisements, local churches, and universities. STATISTICAL ANALYSIS: Analysis of variance and least significant differences were calculated for mean percentage estimation error. Frequencies and Pearson correlation coefficients were used to determine percentage misestimations and relationships between estimations and perceived healthfulness of the food and confidence in the estimate made. RESULTS: In general, accuracy of reporting food amounts was not significantly different for the different types of aids or for the interview technique. Participants frequently overestimated the intake by more than 20% for solids (four overestimations and zero underestimations) and liquids (13 overestimations and one underestimation), and both over- and underestimated amorphous (mounding) foods by more than 20% (six underestimations and seven overestimations). Misestimation was unrelated to perceived healthfulness of the food or respondent confidence in the estimate. CONCLUSIONS: In this study, portion-size data, based on PSEAs used in dietary recalls for estimating amounts eaten, were useful for measuring dietary intake for population-based studies. Individual respondent data, however, may be inaccurate and should be supplemented with additional information to improve assessment of individual nutritional status. Techniques such as telephone interviewing and guiding respondents to certain aids seem useful for reducing the burden of dietary assessment.

Dietary behaviors associated with total fat and saturated fat intake.

OBJECTIVE: To estimate percentages of US adults who have adopted behaviors promoted by dietary guidance about how to reduce fat intake, and to assess relationships between these behaviors and intake of energy from total and saturated fat. DESIGN: Relationships were examined between intake of total and saturated fat from two 24-hour recalls in the US Department of Agriculture's 1994-1996 Continuing Survey of Food Intakes by Individuals and responses to 19 fat-related behavior questions on the follow-up Diet and Health Knowledge Survey (DHKS). SUBJECTS: Data are from a national sample of 5,649 individuals 20 years of age and older. STATISTICAL ANALYSES PERFORMED: Multiple regression models are used to identify dietary behaviors, demographic factors, and personal characteristics that are determinants of fat intake. RESULTS: In this study, the percentage of US adults who consistently followed the low-fat behaviors ranged from 8% to 70%. The most highly adopted behaviors (45% or more of adults) included trimming fat from meat, removing skin from chicken, and eating chips infrequently. The least highly adopted behaviors (15% or less of adults) included eating baked or boiled potatoes without added fat, avoiding butter or margarine on breads, eating low-fat instead of regular cheeses, and having fruit for dessert when dessert is eaten. Together, the 19 fat-related behavior questions on the DHKS formed a statistically significant predictor for total fat and saturated fat intake, expressed as a percent of energy (P<.0001). Key behaviors in terms of their predicted effect on lowering both total and saturated fat intake were never adding fat to baked or boiled potatoes, not eating red meats, eating less than 3 eggs per week, and never eating chicken fried. Predicted effects of these key behaviors in terms of lowering fat intake as a percentage of energy were > or = 1.5 percentage points for total fat and > or = 0.5 percentage point for saturated fat. CONCLUSIONS: Results have applications for designing brief fat assessment instruments and for identifying key nutrition education messages that promote important fat-lowering behaviors.