Exome-Wide Association Study of Pancreatic Cancer Risk.

We conducted a case-control exome-wide association study to discover germline variants in coding regions that affect risk for pancreatic cancer, combining data from 5 studies. We analyzed exome and genome sequencing data from 437 patients with pancreatic cancer (cases) and 1922 individuals not known to have cancer (controls). In the primary analysis, BRCA2 had the strongest enrichment for rare inactivating variants (17/437 cases vs 3/1922 controls) (P = 3.27x10-6; exome-wide statistical significance threshold P < 2.5x10-6). Cases had more rare inactivating variants in DNA repair genes than controls, even after excluding 13 genes known to predispose to pancreatic cancer (adjusted odds ratio, 1.35; P = .045). At the suggestive threshold (P < .001), 6 genes were enriched for rare damaging variants (UHMK1, AP1G2, DNTA, CHST6, FGFR3, and EPHA1) and 7 genes had associations with pancreatic cancer risk, based on the sequence-kernel association test. We confirmed variants in BRCA2 as the most common high-penetrant genetic factor associated with pancreatic cancer and we also identified candidate pancreatic cancer genes. Large collaborations and novel approaches are needed to overcome the genetic heterogeneity of pancreatic cancer predisposition.

Children's school-breakfast reports and school-lunch reports (in 24-h dietary recalls): conventional and reporting-error-sensitive measures show inconsistent accuracy results for retention interval and breakfast location.

Validation-study data were analysed to investigate retention interval (RI) and prompt effects on the accuracy of fourth-grade children's reports of school-breakfast and school-lunch (in 24-h recalls), and the accuracy of school-breakfast reports by breakfast location (classroom; cafeteria). Randomly selected fourth-grade children at ten schools in four districts were observed eating school-provided breakfast and lunch, and were interviewed under one of eight conditions created by crossing two RIs ('short'--prior-24-hour recall obtained in the afternoon and 'long'--previous-day recall obtained in the morning) with four prompts ('forward'--distant to recent, 'meal name'--breakfast, etc., 'open'--no instructions, and 'reverse'--recent to distant). Each condition had sixty children (half were girls). Of 480 children, 355 and 409 reported meals satisfying criteria for reports of school-breakfast and school-lunch, respectively. For breakfast and lunch separately, a conventional measure--report rate--and reporting-error-sensitive measures--correspondence rate and inflation ratio--were calculated for energy per meal-reporting child. Correspondence rate and inflation ratio--but not report rate--showed better accuracy for school-breakfast and school-lunch reports with the short RI than with the long RI; this pattern was not found for some prompts for each sex. Correspondence rate and inflation ratio showed better school-breakfast report accuracy for the classroom than for cafeteria location for each prompt, but report rate showed the opposite. For each RI, correspondence rate and inflation ratio showed better accuracy for lunch than for breakfast, but report rate showed the opposite. When choosing RI and prompts for recalls, researchers and practitioners should select a short RI to maximise accuracy. Recommendations for prompt selections are less clear. As report rates distort validation-study accuracy conclusions, reporting-error-sensitive measures are recommended.

Effectiveness of Prompts on Fourth-Grade Children's Dietary Recall Accuracy Depends on Retention Interval and Varies by Gender.

BACKGROUND: Dietary recall accuracy is related to retention interval (RI) (i.e., time between to-be-reported meals and the interview), and possibly to prompts. To the best of our knowledge, no study has evaluated their combined effect. OBJECTIVE: The combined influence of RI and prompts on children's recall accuracy was investigated in this study. Two RIs [short (prior-24-h recall obtained in afternoon) and long (previous-day recall obtained in morning)] were crossed with 4 prompts [forward (distant-to-recent), meal-name (breakfast, lunch, etc.), open (no instructions), and reverse (recent-to-distant)], creating 8 conditions. METHODS: Fourth-grade children (n = 480; 50% girls) were randomly selected from consenting children at 10 schools in 4 districts in a southern state during 3 school years (2011-2012, 2012-2013, and 2013-2014). Each child was observed eating school-provided breakfast and lunch, and interviewed one time under 1 of the 8 conditions. Condition assignment was constrained so that each had 60 children (30 girls). Accuracy measures were food-item omission and intrusion rates, and energy correspondence rate and inflation ratio. For each measure, linear models determined effects of RI, prompt, gender, and interactions (2-way, 3-way); race/ethnicity, school year, and district were control variables. RESULTS: RI (P values < 0.015) and prompt (P values < 0.005) were significant for all 4 accuracy measures. RI × prompt (P values < 0.001) was significant for 3 accuracy measures (not intrusion rate). Prompt × gender (P = 0.005) was significant for omission rate. RI × prompt × gender was significant for intrusion rate and inflation ratio (P values < 0.001). For the short vs. long RI across prompts and genders, accuracy was better by 33-50% for each accuracy measure. CONCLUSIONS: To obtain the most accurate recalls possible from children, studies should be designed to use a short rather than long RI. Prompts affect children's recall accuracy, although the effectiveness of different prompts depends on RI and varies by gender: at a short RI, the choice of prompts has little systematic effect on accuracy, whereas at a long RI, reverse prompts may elicit the most accurate recalls.

Children's Social Desirability: Effects of Test Assessment Mode.

This study examined a recently developed short version of the Children's Social Desirability (CSD-S) scale with 157 fourth-grade children. Of interest was a) whether one-month test-retest reliability would vary as a function of test assessment mode (interview or classroom), gender, race, SES, and BMI percentile, and b) whether the degree of social desirability would vary as a function of these same variables. The CSD-S scale showed good test-retest reliability for both interview and classroom assessment modes (.85 and .83, respectively). Internal consistency also was good (first interview administration = .84; first classroom administration = .81). Reliability was good and did not vary significantly over assessment mode or any child subgroup variables, suggesting that the CSD-S scale is appropriate for general use. The interview mode elicited significantly more socially desirable answers than did the classroom mode. Social desirability did not differ across child subgroups. Some of these findings were examined, and replicated, on another sample. Thus, the CSD-S scale may be used with diverse groups of children to a) reliably assess a social desirability bias that may systematically bias other self-reports of interest to researchers and b) examine individual differences in degree of social desirability.

Rigid spin-labeled nucleoside C: a nonperturbing EPR probe of nucleic acid conformation.

Rigid spin-labeled nucleoside C, an analog of deoxycytidine that base-pairs with deoxyguanosine, was incorporated into DNA oligomers by chemical synthesis. Thermal denaturation experiments and circular dichroism (CD) measurements showed that C has a negligible effect on DNA duplex stability and conformation. Nucleoside C was incorporated into several positions within single-stranded DNA oligomers that can adopt two hairpin conformations of similar energy, each of which contains a four-base loop. The relative mobility of nucleotides in the alternating C/G hairpin loops, 5'-d(GCGC) and 5'-d(CGCG), was determined by electron paramagnetic resonance (EPR) spectroscopy. The most mobile nucleotide in the loop is the second one from the 5'-end, followed by the third, first and fourth nucleotides, consistent with previous NMR studies of DNA hairpin loops of different sequences. The EPR hairpin data were also corroborated by fluorescence spectroscopy using oligomers containing reduced C (C(f)), which is fluorescent. Furthermore, EPR spectra of duplex DNAs that contained C at the end of the helix showed features that indicated dipolar coupling between two spins. These data are consistent with end-to-end duplex stacking in solution, which was only observed when G was paired to C, but not when C was paired with A, C or T.

Conformational equilibria of bulged sites in duplex DNA studied by EPR spectroscopy.

Conformational flexibility in nucleic acids provides a basis for complex structures, binding, and signaling. One-base bulges directly neighboring single-base mismatches in nucleic acids can be present in a minimum of two distinct conformations, complicating the examination of the thermodynamics by calorimetry or UV-monitored melting techniques. To provide additional information about such structures, we demonstrate how electron paramagnetic resonance (EPR) active spin-labeled base analogues, base-specifically incorporated into the DNA, are monitors of the superposition of different bulge-mismatch conformations. EPR spectra provide information about the dynamic environments of the probe. This information is cast in terms of "dynamic signatures" that have an underlying basis in structural variations. By examining the changes in the equilibrium of the different states across a range of temperatures, the enthalpy and entropy of the interconversion among possible conformations can be determined. The DNA constructs with a single bulge neighboring a single-base mismatch ("bulge-mismatches") may be approximately modeled as an equilibrium between two possible conformations. This structural information provides insight into the local composition of the bulge-mismatch sequences. Experiments on the bulge-mismatches show that basepairing across the helix can be understood in terms of purine and pyrimidine interactions, rather than specific bases. Measurements of the enthalpy and entropy of formation for the bulge-mismatches by differential scanning calorimetry and UV-monitored melting confirm that the formation of bulge-mismatches is in fact more complicated than a simple two-state process, consistent with the base-specific spectral data that bulge-mismatches exist in multiple conformations in the premelting temperature region. We find that the calculations with the nearest-neighbor (NN) model for the two likely conformations do not correlate well with the populations of structures and thermodynamic parameters inferred from the base-specific EPR dynamics probe. We report that the base-specific spin probes are able to identify a bistable, temperature dependent, switching between conformations for a particular complex bulged construct.

Integration of Genomic and Transcriptional Features in Pancreatic Cancer Reveals Increased Cell Cycle Progression in Metastases.

We integrated clinical, genomic, and transcriptomic data from 224 primaries and 95 metastases from 289 patients to characterize progression of pancreatic ductal adenocarcinoma (PDAC). Driver gene alterations and mutational and expression-based signatures were preserved, with truncations, inversions, and translocations most conserved. Cell cycle progression (CCP) increased with sequential inactivation of tumor suppressors, yet remained higher in metastases, perhaps driven by cell cycle regulatory gene variants. Half of the cases were hypoxic by expression markers, overlapping with molecular subtypes. Paired tumor heterogeneity showed cancer cell migration by Halstedian progression. Multiple PDACs arising synchronously and metachronously in the same pancreas were actually intra-parenchymal metastases, not independent primary tumors. Established clinical co-variates dominated survival analyses, although CCP and hypoxia may inform clinical practice.

Theory for spin-lattice relaxation of spin probes on weakly deformable DNA.

The weakly bending rod (WBR) model of double-stranded DNA (dsDNA) is adapted to analyze the internal dynamics of dsDNA as observed in electron paramagnetic resonance (EPR) measurements of the spin-lattice relaxation rate, R(1e), for spin probes rigidly attached to nucleic acid-bases. The WBR theory developed in this work models dsDNA base-pairs as diffusing rigid cylindrical discs connected by bending and twisting springs whose elastic force constants are kappa and alpha, respectively. Angular correlation functions for both rotational displacement and velocity are developed in detail so as to compute values for R(1e) due to four relaxation mechanisms: the chemical shift anisotropy (CSA), the electron-nuclear dipolar (END), the spin rotation (SR), and the generalized spin diffusion (GSD) relaxation processes. Measured spin-lattice relaxation rates in dsDNA under 50 bp in length are much faster than those calculated for the same DNAs modeled as rigid rods. The simplest way to account for this difference is by allowing for internal flexibility in models of DNA. Because of this discrepancy, we derive expressions for the spectral densities due to CSA, END, and SR mechanisms directly from a weakly bending rod model for DNA. Special emphasis in this development is given to the SR mechanism because of the lack of such detail in previous treatments. The theory developed in this paper provides a framework for computing relaxation rates from the WBR model to compare with magnetic resonance relaxation data and to ascertain the twisting and bending force constants that characterize DNA.

The National Health and Nutrition Examination Survey's Food Insecurity Questionnaire Completed by Children: Effects of Assessment Mode (Classroom versus Interview).

The National Health and Nutrition Examination Survey's food insecurity questionnaire was administered to 155 children (77 African American, 65 White, 13 "Other" [7 Hispanic; 6 mixed races]) in grade 4 twice, 28-32 days apart. Test-retest reliabilities were modest and somewhat similar for assessment mode (classroom, interview) and subgroup variables (gender, race, socioeconomic status, academic achievement, body mass index percentile, social desirability). As academic achievement increased, White and Other children reported less food insecurity, and African-American children reported slightly less. As social desirability increased, White and African-American children reported slightly more food insecurity, and Other children reported substantially more. Although the questionnaire may be acceptable for use with diverse groups of children in grade 4, validation is needed.

Reflex Testing for Germline BRCA1, BRCA2, PALB2, and ATM Mutations in Pancreatic Cancer: Mutation Prevalence and Clinical Outcomes From Two Canadian Research Registries.

PURPOSE: We investigated the translational value of reflex testing for germline mutations in four homology-directed DNA repair predisposition genes (BRCA1, BRCA2, PALB2, and ATM) in consecutive patients with pancreatic adenocarcinoma. METHODS: One hundred fifty patients with French-Canadian (FC) ancestry were evaluated for founder mutations, and 114 patients were subsequently assessed by full gene sequencing and multiplex ligation-dependent probe amplification for nonfounder mutations. Two hundred thirty-six patients unselected for ancestry were also assessed for mutations by full gene sequencing. RESULTS: The FC founder mutation prevalence among the 150 patients was 5.3% (95% CI, 2.6% to 10.3%), and the nonfounder mutation prevalence across the four genes among the 114 patients tested was 2.6% (95% CI, 0.6% to 7.8%). In the case series unselected for ancestry, 10.0% (95% CI, 2.7% to 26.4%) of patients reporting Ashkenazi Jewish (AJ) ancestry carried an AJ founder mutation, with no nonfounder mutations identified. The mutation prevalence among patients without FC/AJ ancestry was 4.9% (95% CI, 2.6% to 8.8%). Mutations were more frequent in patients diagnosed at ≤ 50 years of age (P = .03) and in patients with either two or more first- or second-degree relatives with pancreas, breast, ovarian or prostate cancer, or one such relative and a second primary of one of these cancer types (P < .001). BRCA1, BRCA2, and PALB2 carriers with late-stage (III or IV) disease had an overall survival advantage (P = .049), particularly if treated with platinum-based chemotherapies (P = .030). CONCLUSION: Considering these results, we recommend reflex founder mutation testing of patients with FC/AJ ancestry and full gene sequencing of patients who are ≤ 50 years or meet the identified family history criteria. Reflex testing of all incident patients for these four genes may become justified as full gene sequencing costs decline.

Candidate DNA repair susceptibility genes identified by exome sequencing in high-risk pancreatic cancer.

The genetic basis underlying the majority of hereditary pancreatic adenocarcinoma (PC) is unknown. Since DNA repair genes are widely implicated in gastrointestinal malignancies, including PC, we hypothesized that there are novel DNA repair PC susceptibility genes. As germline DNA repair gene mutations may lead to PC subtypes with selective therapeutic responses, we also hypothesized that there is an overall survival (OS) difference in mutation carriers versus non-carriers. We therefore interrogated the germline exomes of 109 high-risk PC cases for rare protein-truncating variants (PTVs) in 513 putative DNA repair genes. We identified PTVs in 41 novel genes among 36 kindred. Additional genetic evidence for causality was obtained for 17 genes, with FAN1, NEK1 and RHNO1 emerging as the strongest candidates. An OS difference was observed for carriers versus non-carriers of PTVs with early stage (≤IIB) disease. This adverse survival trend in carriers with early stage disease was also observed in an independent series of 130 PC cases. We identified candidate DNA repair PC susceptibility genes and suggest that carriers of a germline PTV in a DNA repair gene with early stage disease have worse survival.

Whole Genome Sequencing Defines the Genetic Heterogeneity of Familial Pancreatic Cancer.

UNLABELLED: Pancreatic cancer is projected to become the second leading cause of cancer-related death in the United States by 2020. A familial aggregation of pancreatic cancer has been established, but the cause of this aggregation in most families is unknown. To determine the genetic basis of susceptibility in these families, we sequenced the germline genomes of 638 patients with familial pancreatic cancer and the tumor exomes of 39 familial pancreatic adenocarcinomas. Our analyses support the role of previously identified familial pancreatic cancer susceptibility genes such as BRCA2, CDKN2A, and ATM, and identify novel candidate genes harboring rare, deleterious germline variants for further characterization. We also show how somatic point mutations that occur during hematopoiesis can affect the interpretation of genome-wide studies of hereditary traits. Our observations have important implications for the etiology of pancreatic cancer and for the identification of susceptibility genes in other common cancer types. SIGNIFICANCE: The genetic basis of disease susceptibility in the majority of patients with familial pancreatic cancer is unknown. We whole genome sequenced 638 patients with familial pancreatic cancer and demonstrate that the genetic underpinning of inherited pancreatic cancer is highly heterogeneous. This has significant implications for the management of patients with familial pancreatic cancer.

Increased in vitro and in vivo sensitivity of BRCA2-associated pancreatic cancer to the poly(ADP-ribose) polymerase-1/2 inhibitor BMN 673.

BRCA2-associated pancreatic ductal adenocarcinoma (PDAC) may be sensitive to agents that target homology-directed DNA repair, such as DNA crosslinking agents (DCLs) and PARP inhibitors (PARPis). Here, we assessed the sensitivities of BRCA2-deficient (Capan-1) and BRCA2-proficient (MIA PaCa-2) PDAC cell lines to a panel of DCLs and PARPis. Compared to MIA PaCa-2, Capan-1 was significantly more sensitive to all tested DCLs and PARPis, with similar increased sensitivities to cisplatin and the PARPi BMN 673 compared to other DCLs and the PARPi veliparib. We provide further support for this observation by showing that shRNA-mediated BRCA2 knockdown in PANC-1, a BRCA2-proficient cell line, induces sensitization to cisplatin and BMN 673 but not to veliparib. These findings were validated in a PDAC murine xenograft model derived from a patient with bi-allelic BRCA2 mutations. We found 64% and 61% tumor growth inhibition of this xenograft with cisplatin and BMN 673 treatments, respectively. Cisplatin and BMN 673 treatments reduced cellular proliferation and induced apoptosis. Our findings support a personalized treatment approach for BRCA2-associated PDAC.

Room-temperature electron spin dynamics in free-standing ZnO quantum dots.

Conduction band electrons in colloidal ZnO quantum dots have been prepared photochemically and examined by electron paramagnetic resonance spectroscopy. Nanocrystals of 4.6 nm diameter containing single S-shell conduction band electrons have g(*)=1.962 and a room-temperature ensemble spin-dephasing time of T(2)(*)=25 ns, as determined from linewidth analysis. Increasing the electron population leads to increased g(*) and decreased T(2)(*), both associated with formation of P-shell configurations. A clear relationship between T(2)(*) and hyperfine coupling with 67Zn(I=5/2) is observed.

Membrane surface dynamics of DNA-threaded nanopores revealed by simultaneous single-molecule optical and ensemble electrical recording.

We describe a method for simultaneous single-molecule optical and electrical characterization of membrane-based sensors that contain ion-channel nanopores. The technique is used to study the specific and nonspecific interactions of streptavidin-capped DNA polymers with lipid bilayers composed of diphytanoyl phosphatidylcholine and diphytanoyl phosphatidylglycerol. Biotinylated DNA that is bound to fluorescently labeled streptavidin is electrophoretically driven into, or away from, the lumen of alpha hemolysin (alphaHL) ion channels by an external electric field. Confocal microscopy simultaneously captures single-molecule fluorescence dynamics from the membrane interface at different applied potentials. Fluorescence correlation analysis is used to determine the surface number density and diffusion constant of membrane-associated complexes. The dual optical and electrical approach can detect membrane-associated species at a surface coverage below 10(-5) monolayers of streptavidin, a sensitivity that surpasses most other in vitro surface analysis techniques. By comparing the change in transmembrane current to the number of fluorescent molecules leaving the bilayer when the electrical potential is reversed, we demonstrate the general utility of the approach within the context of nanopore-based sensing and discuss a mechanism by which DNA-streptavidin complexes can be nonspecifically retained at the membrane interface.