Multiple independent genetic factors at NOS1AP modulate the QT interval in a multi-ethnic population.

Extremes of electrocardiographic QT interval are associated with increased risk for sudden cardiac death (SCD); thus, identification and characterization of genetic variants that modulate QT interval may elucidate the underlying etiology of SCD. Previous studies have revealed an association between a common genetic variant in NOS1AP and QT interval in populations of European ancestry, but this finding has not been extended to other ethnic populations. We sought to characterize the effects of NOS1AP genetic variants on QT interval in the multi-ethnic population-based Dallas Heart Study (DHS, n = 3,072). The SNP most strongly associated with QT interval in previous samples of European ancestry, rs16847548, was the most strongly associated in White (P = 0.005) and Black (P = 3.6 x 10(-5)) participants, with the same direction of effect in Hispanics (P = 0.17), and further showed a significant SNP x sex-interaction (P = 0.03). A second SNP, rs16856785, uncorrelated with rs16847548, was also associated with QT interval in Blacks (P = 0.01), with qualitatively similar results in Whites and Hispanics. In a previously genotyped cohort of 14,107 White individuals drawn from the combined Atherosclerotic Risk in Communities (ARIC) and Cardiovascular Health Study (CHS) cohorts, we validated both the second locus at rs16856785 (P = 7.63 x 10(-8)), as well as the sex-interaction with rs16847548 (P = 8.68 x 10(-6)). These data extend the association of genetic variants in NOS1AP with QT interval to a Black population, with similar trends, though not statistically significant at P<0.05, in Hispanics. In addition, we identify a strong sex-interaction and the presence of a second independent site within NOS1AP associated with the QT interval. These results highlight the consistent and complex role of NOS1AP genetic variants in modulating QT interval.

THE RELATIONSHIP BETWEEN OBESITY AND PSYCHOLOGICAL GENERAL WELL-BEING IN UNITED STATES WOMEN

While clinical studies have shown a negative relationship between obesity and mental health in women, population studies have not shown a consistent association. However, many of these studies can be criticized regarding fatness level criteria, lack of control variables, and validity of the psychological variables.^ The purpose of this research was to elucidate the relationship between fatness level and mental health in United States women using data from the First National Health and Nutrition Examination Survey (NHANES I), which was conducted on a national probability sample from 1971 to 1974. Mental health was measured by the General Well-Being Schedule (GWB), and fatness level was determined by the sum of the triceps and subscapular skinfolds. Women were categorized as lean (15th percentile or less), normal (16th to 84th percentiles), or obese (85th percentile or greater).^ A conceptual framework was developed which identified the variables of age, race, marital status, socioeconomic status (education), employment status, number of births, physical health, weight history, and perception of body image as important to the fatness level-GWB relationship. Multiple regression analyses were performed separately for whites and blacks with GWB as the response variable, and fatness level, age, education, employment status, number of births, marital status, and health perception as predictor variables. In addition, 2- and 3-way interaction terms for leanness, obesity and age were included as predictor variables. Variables related to weight history and perception of body image were not collected in NHANES I, and thus were not included in this study.^ The results indicated that obesity was a statistically significant predictor of lower GWB in white women even when the other predictor variables were controlled. The full regression model identified the young, more educated, obese female as a subgroup with lower GWB, especially in blacks. These findings were not consistent with the previous non-clinical studies which found that obesity was associated with better mental health. The social stigma of being obese and the preoccupation of women with being lean may have contributed to the lower GWB in these women. ^

Multiple imputation for missing continuous and dichotomous data: The effects of patterns of missingness and variable correlations on type I error with small samples

The purpose of this study is to investigate the effects of predictor variable correlations and patterns of missingness with dichotomous and/or continuous data in small samples when missing data is multiply imputed. Missing data of predictor variables is multiply imputed under three different multivariate models: the multivariate normal model for continuous data, the multinomial model for dichotomous data and the general location model for mixed dichotomous and continuous data. Subsequent to the multiple imputation process, Type I error rates of the regression coefficients obtained with logistic regression analysis are estimated under various conditions of correlation structure, sample size, type of data and patterns of missing data. The distributional properties of average mean, variance and correlations among the predictor variables are assessed after the multiple imputation process. ^ For continuous predictor data under the multivariate normal model, Type I error rates are generally within the nominal values with samples of size n = 100. Smaller samples of size n = 50 resulted in more conservative estimates (i.e., lower than the nominal value). Correlation and variance estimates of the original data are retained after multiple imputation with less than 50% missing continuous predictor data. For dichotomous predictor data under the multinomial model, Type I error rates are generally conservative, which in part is due to the sparseness of the data. The correlation structure for the predictor variables is not well retained on multiply-imputed data from small samples with more than 50% missing data with this model. For mixed continuous and dichotomous predictor data, the results are similar to those found under the multivariate normal model for continuous data and under the multinomial model for dichotomous data. With all data types, a fully-observed variable included with variables subject to missingness in the multiple imputation process and subsequent statistical analysis provided liberal (larger than nominal values) Type I error rates under a specific pattern of missing data. It is suggested that future studies focus on the effects of multiple imputation in multivariate settings with more realistic data characteristics and a variety of multivariate analyses, assessing both Type I error and power. ^

Obesity and risk of multiple myeloma: A case-control study

Introduction. Several studies have reported a positive association of body mass index (BMI) with multiple myeloma; however, the period of adulthood where BMI is most important remains unclear. In addition, it is well known that body fat is associated with both sex-steroid hormone storage and with increasing insulin levels; therefore, it was hypothesized that the association between obesity and multiple myeloma may be attributed to increased aromatization of androgen in adipose tissue. Objective. The overall objective of this case-control study was to determine whether multiple myeloma cases had higher BMI and greater adult weight gain relative to healthy controls. In addition, we tested the hypothesis that hormone replacement therapy use among women will further increase the association between BMI and risk of multiple myeloma. This study used data from a pilot case-control study at M.D. Anderson Cancer Center (MDACC), entitled Etiology of multiple myeloma, directed by Dr. Sara Strom and Dr. Sergio Giralt. Methods. The pilot study recruited a total of 122 cases of histopathologically confirmed multiple myeloma from MDACC. Controls (n=183) were selected from a database of random digit dialing controls accrued in the Department of Epidemiology at MDACC and were frequency matched to the cases on age (±5 years), gender, and race/ethnicity. Demographic and risk factor information were obtained from all participants who completed a self-administered questionnaire. Items included in the questionnaire include demographic information, height and weight at age 25, 40 and current/diagnosis, medical history, family history of cancer, smoking and alcohol use. Statistical analysis. Initial descriptive analysis included Student's t-test and Pearson's chi-squared tests. Odds ratios and 95% confidence intervals were calculated to quantify the association between the variables of interest and multiple myeloma. A multivariable model will be developed using unconditional logistic regression. Results. MM cases were 1.79 times (95% CI=0.99-3.32) more likely to have been overweight or obese (BMI > 25 kg/m2) at age 25 relative to healthy controls after controlling for age, gender, race/ethnicty, education and family history of cancer. Being overweight or obese at age 40 was not significantly associated with mutliple myeloma risk (OR=1.42, 95% CI=0.86-2.34) nor was being overweight or obses at diagnosis (OR=1.43, 95% CI=0.78, 2.63). We observed a statistically significant 2-fold increased odds of multiple myeloma in individuals who gained more than 4.7 kg during between 25 and 40 years (OR=1.97, 95% CI=1.15-3.39). When assessing HRT as a modifier of the BMI and multiple myeloma association among women (N=123), no association between obesity and MM status was observed among women who have never used HRT (OR=0.60, 95% CI=0.23-1.61; n=73). Yet among women who have ever used HRT (n=50), being overweight or obese was associated with an increase in MM risk (OR=2. 93, 95% CI=0.81-10.6) after adjusting for age; however, the association was not statistically significant. Significance. This study provides further evidence that increased BMI increases the risk of multiple myeloma. Furthermore, among women, HRT use may modify risk of disease. ^

Safety, pharmacokinetics, and antiretroviral activity of multiple doses of ibalizumab (formerly TNX-355), an anti-CD4 monoclonal antibody in HIV-1 infected adults

Context: Despite tremendous strides in HIV treatment over the past decade, resistance remains a major problem. A growing number of patients develop resistance and require new therapies to suppress viral replication. ^ Objective: To assess the safety of multiple administrations of the anti-CD4 receptor (anti-CD4) monoclonal antibody ibalizumab given as intravenous (IV) infusions, in three dosage regimens, in subjects infected with human immunodeficiency virus (HIV-1). ^ Design: Phase 1, multi-center, open-label, randomized clinical trial comparing the safety, pharmacokinetics and antiviral activity of three dosages of ibalizumab. ^ Setting: Six clinical trial sites in the United States. ^ Participants: A total of twenty-two HIV-positive patients on no anti-retroviral therapy or a stable failing regimen. ^ Intervention: Randomized to one of two treatment groups in Arms A and B followed by non-randomized enrollment in Arm C. Patients randomized to Arm A received 10 mg/kg of ibalizumab every 7 days, for a total of 10 doses; patients randomized to Arm B received a total of six doses of ibalizumab; a single loading dose of 10 mg/kg on Day 1 followed by five maintenance doses of 6 mg/kg every 14 days, starting at Week 1. Patients assigned to Arm C received 25 mg/kg of ibalizumab every 14 days for a total of 5 doses. All patients were followed for safety for an additional 7 to 8 weeks. ^ Main Outcome Measures: Clinical and laboratory assessments of safety and tolerability of multiple administrations of ibalizumab in HIV-infected patients. Secondary measures of efficacy include HIV-1 RNA (viral load) measurements. ^ Results: 21 patients were treatment-experienced and 1 was naïve to HIV therapy. Six patients were failing despite therapy and 15 were on no current HIV treatment. Mean baseline viral load (4.78 log 10; range 3.7-5.9) and CD4+ cell counts (332/μL; range 89-494) were similar across cohorts. Mean peak decreases in viral load from baseline of 0.99 log10(1.11 log10, and 0.96 log 10 occurred by Wk 2 in Cohorts A, B and C, respectively. Viral loads decreased by >1.0 log10 in 64%; 4 patients viral loads were suppressed to < 400 copies/mL. Viral loads returned towards baseline by Week 9 with reduced susceptibility to ibalizumab. CD4+ cell counts rose transiently and returned toward baseline. Maximum median elevations above BL in CD4+ cell counts for Cohorts A, B and C were +257, +198 and +103 cells/μL, respectively and occurred within 3 Wks in 16 of 22 subjects. The half-life of ibalizumab was 3-3.5 days and elimination was characteristic of capacity-limited kinetics. Administration of ibalizumab was well tolerated. Four serious adverse events were reported during the study. None of these events were related to study drug. Headache, nausea and cough were the most frequently reported treatment emergent adverse events and there were no laboratory abnormalities related to study drug. ^ Conclusions: Ibalizumab administered either weekly or bi-weekly was safe, well tolerated, and demonstrated antiviral activity. Further studies with ibalizumab in combination with standard antiretroviral treatments are warranted.^

CARMA3 serves as a critical mediator of NF-kappaB in multiple signal transduction pathways

The central dogma of molecular biology dictates that DNA is transcribed into RNA, which is later translated into protein. One of the early activators in this process is the transcription factor NF-κB. We have determined that an NF-κB inducer, CARMA3, is required for proper neural tube closure, similar to other NF-κB inducers. Using a genetic knockout of CARMA3, we demonstrated that it is required for Gαq-coupled GPCR-induced NF-κB activation. This is facilitated through a MAPK and IKK phosphorylation-independent mechanism, most likely by controlling NEMO-associated ubiquitination. We have also shown that CARMA3 is required for EGF and HRG-induced NF-κB activation. This activation requires the activity of both EGFR and HER2, as well as PKC. Again, we observed no defect in IKK phosphorylation, although we determined a clear defect in IKK activation. Finally, we have begun to determine the role of CARMA3 to both EGFR and HER2-induced tumorigenicity. By overexpressing a constitutive active mutant of HER2 in our CARMA3 WT and KO MEF cells, we have shown CARMA3 is important for HER2-driven soft agar colony growth. We have also shown that knockdown of endogenous CARMA3 in the EGFR-overexpressing A431 cell line abolishes EGF-induced NF-κB activation. These same cells have a dramatically reduced capacity to form colonies in soft agar as well. Using both mouse xenografts and a transgenic model of HER2-induced breast cancer, we have initiated studies which will help to determine the role of CARMA3 to in vivo tumorigenesis. Collectively, this work reveals novel roles for the CARMA3 protein in development, GPCR and EGFR/HER2 signaling. It also suggests that CARMA3 is involved in EGFR/HER2 mediated tumorigenesis, possibly indicating a novel therapeutic target for use in treatment of cancer. ^

The survival role and the potential therapeutic targeting of MET in multiple myeloma

Multiple myeloma (MM) is a debilitating and incurable B-cell malignancy. Previous studies have documented that the hepatocyte growth factor (HGF) plays a role in the pathobiology of MM. The receptor tyrosine kinase MET induced signaling initiates when its ligand HGF binds to the MET receptor. However, the direct importance of MET in MM has not been elucidated. The present work used three different but complementary approaches to reduce MET protein levels or its activity to demonstrate the importance of MET in MM. ^ In the first approach, MET transcript and protein levels were reduced by directly targeting the cellular MET transcripts using shRNA retroviral infection techniques. This direct reduction of MET mRNA leads to a reduction of MET protein levels, which caused an inhibition of growth and induction of cell death. ^ In the second approach, a global transcription inhibitor flavopiridol was used as a potential pharmacological tool to reduce MET levels. MET has a short half-life of 30 min for mRNA and 4 hours for protein; therefore using a RNA pol II inhibitor such as flavopiridol would be a viable option to reduce MET levels. When using flavopiridol in MM cell lines, there was a reduction of MET transcript and protein levels, which was associated with the induction of cell death. ^ Finally in the last strategy, MET kinase activity was suppressed by MP470, a small molecule inhibitor that binds to the ATP binding pocket in the kinase domain. At concentrations where phosphorylation of MET was inhibited there was induction of cell death in MM cell lines and primary cells from patients. In addition, in MM cell lines there was a decrease in phosphorylation of AKT (ser473) and caspase-9 (ser196); downstream of MET, suggesting that the mechanism of action for survival may be through these cascade of events. ^ Overall, this study provides a proof-of-principle that MET is important for the survival of MM cell lines as well as primary plasma cells obtained from patients. Therefore, targeting MET therapeutically may be a possible strategy to treat patients with this debilitating disease of MM. ^

Statistical Methods for Differential Expressions of Genes Detected in Multiple-Condition Experiment of Microarray

Most studies of differential gene-expressions have been conducted between two given conditions. The two-condition experimental (TCE) approach is simple in that all genes detected display a common differential expression pattern responsive to a common two-condition difference. Therefore, the genes that are differentially expressed under the other conditions other than the given two conditions are undetectable with the TCE approach. In order to address the problem, we propose a new approach called multiple-condition experiment (MCE) without replication and develop corresponding statistical methods including inference of pairs of conditions for genes, new t-statistics, and a generalized multiple-testing method for any multiple-testing procedure via a control parameter C. We applied these statistical methods to analyze our real MCE data from breast cancer cell lines and found that 85 percent of gene-expression variations were caused by genotypic effects and genotype-ANAX1 overexpression interactions, which agrees well with our expected results. We also applied our methods to the adenoma dataset of Notterman et al. and identified 93 differentially expressed genes that could not be found in TCE. The MCE approach is a conceptual breakthrough in many aspects: (a) many conditions of interests can be conducted simultaneously; (b) study of association between differential expressions of genes and conditions becomes easy; (c) it can provide more precise information for molecular classification and diagnosis of tumors; (d) it can save lot of experimental resources and time for investigators.^

Motivational interviewing and feedback for college drinkers: Handling missing values with complete case analysis and multiple imputation

Objective: In this secondary data analysis, three statistical methodologies were implemented to handle cases with missing data in a motivational interviewing and feedback study. The aim was to evaluate the impact that these methodologies have on the data analysis. ^ Methods: We first evaluated whether the assumption of missing completely at random held for this study. We then proceeded to conduct a secondary data analysis using a mixed linear model to handle missing data with three methodologies (a) complete case analysis, (b) multiple imputation with explicit model containing outcome variables, time, and the interaction of time and treatment, and (c) multiple imputation with explicit model containing outcome variables, time, the interaction of time and treatment, and additional covariates (e.g., age, gender, smoke, years in school, marital status, housing, race/ethnicity, and if participants play on athletic team). Several comparisons were conducted including the following ones: 1) the motivation interviewing with feedback group (MIF) vs. the assessment only group (AO), the motivation interviewing group (MIO) vs. AO, and the intervention of the feedback only group (FBO) vs. AO, 2) MIF vs. FBO, and 3) MIF vs. MIO.^ Results: We first evaluated the patterns of missingness in this study, which indicated that about 13% of participants showed monotone missing patterns, and about 3.5% showed non-monotone missing patterns. Then we evaluated the assumption of missing completely at random by Little's missing completely at random (MCAR) test, in which the Chi-Square test statistic was 167.8 with 125 degrees of freedom, and its associated p-value was p=0.006, which indicated that the data could not be assumed to be missing completely at random. After that, we compared if the three different strategies reached the same results. For the comparison between MIF and AO as well as the comparison between MIF and FBO, only the multiple imputation with additional covariates by uncongenial and congenial models reached different results. For the comparison between MIF and MIO, all the methodologies for handling missing values obtained different results. ^ Discussions: The study indicated that, first, missingness was crucial in this study. Second, to understand the assumptions of the model was important since we could not identify if the data were missing at random or missing not at random. Therefore, future researches should focus on exploring more sensitivity analyses under missing not at random assumption.^

Activation of matrix metalloproteinase -9 (MMP-9) by heregulin in human breast cancer cells involves multiple signaling pathways

Matrix metalloproteinase-9 (MMP-9) plays an important role in tumor invasion and angiogenesis. Secretion of MMP-9 has been reported in various cancer types including lung cancer, brain cancer, colon cancer, and breast cancer. Heregulin is a growth factor that regulates growth and differentiation of normal breast cells as well as mammary tumor cells. To study the role of heregulin in breast cancer metastasis, we tested whether heregulin may regulate MMP-9 secretion. By screening a panel of breast cancer cell line for their ability to respond to heregulin and produce MMP-9, we have found that MMP-9 secretion can be induced by heregulin-β1 in two breast cancer cell lines, SKBr3 and MCF-7. In both cell lines, increase of MMP-9 activity as shown by zymography was accompanied by increased protein level as well as mRNA level of MMP-9. Using a reporter luciferase assay, we have identified that proximal −670bp promoter of MMP-9 had similar activity to a 2.2kb MMP-9 promoter in response to heregulin stimulation. Heregulin treatment of SKBr3 and MCF-7 activated multiple signaling pathways inside cells. These include the Erk pathway, the p38 kinase pathway, PKC pathway, and PI-3K pathway. To examine which pathways are involved in MMP-9 activation by heregulin, we have used a panel of chemical inhibitors to specifically inhibit each one of these pathways. Ro-31-8220 (PKC inhibitor) and SB203580 (p38 kinase inhibitor) completely blocked heregulin activation of MMP-9. On the other hand, PD098059 (MEK-1 inhibitor) partially blocked MMP-9 activation, whereas PI-3K inhibitor, wortmannin, had no effect. Therefore, at least three signaling pathways are involved in activation of MMP-9 by heregulin. Since MMP-9 is tightly associated with metastatic potential, our study also suggests that heregulin may enhance breast tumor metastasis through induction of MMP-9 expression. ^

Genome instability quantified in constitutive tissues as well as putatively stable tumor tissue in hereditary non -polyposis colon cancer patients by small pool PCR

Microsatellite instability (MSI) is a hallmark of the mutator phenotype associated with Hereditary Non-Polyposis Colon Cancer (HNPCC). The MSI-High (MSI-H) HNPCC population has been well characterized, but the microsatellite low and stable (MSI-L/MSS) HNPCC population is much less understood. We hypothesize there are significant levels of MSI in HNPCC DNA classified as MSI-L/MSS, but no single variant allele makes up a sufficient population in the tumor DNA to be detected by standard analysis. Finding variants would suggest there is a mutator phenotype for the MSI-L/MSS HNPCC population that is distinct from the MSI-H HNPCC populations. This study quantified and compared MSI in HNPCC patients previously shown to be MSI-H, MSI-L/MSS and an MSI-H older, sporadic colorectal cancer patient. Small-pool Polymerase Chain Reactions (SP-PCRs) were conducted where the DNAs from each sample and controls are diluted into multiple pools, each containing approximately single genome equivalents. At least 100 alleles/sample were studied at six microsatellite loci. Mutant fragments were identified, quantified, and compared using Poisson statistics. Most of the variants were small deletions or insertions, with more mutants being deletions, as has been previously described in yeast and transgenic mice. SP-PCR, where most of the pools contained only 3 or less fragments, enabled identification of variants too infrequent to be detected by large pool PCR. Mutant fragments in positive control MSI-H tumor samples ranged from 0.26 to 0.68 in at least 4 of the 6 loci tested and were consistent with their MSI-H status. In the so called MSS tumors and constitutive tissues (normal colon tissue, and PBLs) of all the HNPCC patients, low, but significant levels of MSI were seen in at least two of the loci studied. This phenomenon was not seen in the sporadic MSI constitutive tissues nor the normal controls and suggests haploinsufficiency, gain-of-function, or a dominant/negative basis of the instability in HNPCC patients carrying germline mutations for tumor suppressor genes. A different frequency and spectrum of mutant fragments suggests a different genetic basis (other than a major mutation in MLH1 or MSH2) for disease in MSI-L and MSS HNPCC patients. ^