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. ^

Structure-function analyses of {\it PAX6\/} and the molecular bases of aniridia

PAX6 is a transcription activator that regulates eye development in animals ranging from Drosophila to human. The C-terminal region of PAX6 is proline/serine/threonine-rich (PST) and functions as a potent transactivation domain when attached to a heterologous DNA-binding domain of the yeast transcription factor, GAL4. The PST region comprises 152 amino acids encoded by four exons. The transactivation function of the PST region has not been defined and characterized in detail by in vitro mutagenesis. I dissected the PST domain in two independent systems, a heterologous system using a GAL4 DNA-binding site and the native system of PAX6. In both systems, the results show consistently that all four constituent exons of the PST domain are responsible for the transactivation function. The four exon fragments act cooperatively to stimulate transcription, although none of them can function individually as an independent transactivation domain. Combinations of two or more exon fragments can reconstitute substantial transactivation activity when fused to the DNA-binding domain of GAL4, but they surprisingly do not produce much activity in the context of native PAX6 even though the mutant PAX6 proteins are stable and their DNA-binding function remains unaffected. I conclude that the PAX6 protein contains an unusually large transactivation domain that is evolutionarily conserved to a high degree, and that its full transactivation activity relies on the cooperative action of the four exon fragments.^ Most PAX6 mutations detected in patients with aniridia result in truncations of the protein. Some of the truncation mutations occur in the PST region of PAX6, resulting in mutant proteins that retain their DNA-binding ability but have no significant transactivation activity. It is not clear whether such mutants are true loss-of-function or dominant-negative mutants. I show that these mutants are dominant-negative if they are coexpressed with wild-type PAX6 in cultured cells and that the dominant-negative effects result from enhanced DNA-binding ability of these mutants due to removal of the PST domain. These mutants are able to repress the wild-type PAX6 activity not only at target genes with paired domain binding sites but also at target genes with homeodomain binding sites.^ Mutations in the human PAX6 gene produce various phenotypes, including aniridia, Peters' anomaly, autosomal dominant keratitis, and familial foveal dysplasia. The various phenotypes may arise from different mutations in the same gene. To test this theory, I performed a functional analysis of two missense mutations in the paired domain: the R26G mutation reported in a case of Peters' anomaly, and the I87R mutation identified in a patient with aniridia. While both the R26 and the I87 positions are conserved in the paired boxes of all known PAX genes, X-ray crystallography has shown that only R26 makes contact with DNA. I found that the R26G mutant failed to bind a subset of paired domain binding sites but, surprisingly, bound other sites and successfully transactivated promoters containing those sites. In contrast, the I87R mutant had lost the ability to bind DNA at all tested sites and failed to transactivate promoters. My data support the haploinsufficiency hypothesis of aniridia, and the hypothesis that R26G is a hypomorphic allele. ^

Physiologic and psychosocial stage-based differences for dietary fat consumption in women with type 2 diabetes

Purpose. This cross-sectional, observational study explored differences among groups staged for intent to decrease dietary fat intake in women with type 2 diabetes in relation to demographic, weight concern, physiological, and psychosocial variables. ^ Methods. A sample of 100 community-dwelling, English-speaking women, who were over age 30 and had type 2 diabetes for at least a year, was accessed through a culturally diverse endocrinology clinic. Subjects completed 7 self-report instruments: demographic sheet, with 11-point weight satisfaction scale; staging algorithm; fat intake (MEDFICTS); depression (CES-D); diabetes-specific dietary knowledge (ADKnowl), social support and self-efficacy scales (SE-Type 2). Physiological variables were abstracted from the medical record (HbA 1c, blood pressure, serum cholesterol and triglycerides). ^ Results. The women's average age was 57.69 years ( SD = 3.07); 50% were married. Subjects were well-educated ( M = 14 years; SD = 3.33), with average diabetes duration of 10.57 years (SD = 9.11), high body mass index (M = 35.72; SD = 8.36), low diabetes-specific dietary knowledge, low weight satisfaction, but in good diabetes control. Racial/ethnic composition was 44% non-Hispanic-White-American, 18% Hispanic-White-American, 15% non-Hispanic-African-American, 16% Hispanic-African-American and 5% other. Fat intake was low and differed by racial/ethnic demographics. The highest fat intake scores were for non-Hispanic-African-Americans (M = 53), followed by Hispanic-White-Americans (M = 51), non-Hispanic-White-Americans (M = 45), and Hispanic-African-Americans (M = 32), who had the lowest fat intake scores. ^ MANOVA analyses revealed no significant differences between stages of behavior change in relation to psychosocial or weight concern variables, age, education, HbA1c, or cholesterol levels. Single women were more likely to be in the three preaction stages (precontemplation, contemplation, and preparation); married women were equally distributed across stages (the preaction stages plus action and maintenance). African-American women (Hispanic and non-Hispanic) were more likely in contemplation and preparation. Triglycerides were higher in women in the action stage than contemplation or preparation. Systolic blood pressure was higher in action than preparation; diastolic blood pressure was higher in action than preaction. ^ Conclusions. Healthcare professionals should consider race, ethnicity, and marital status in client interactions. Dietary intake can vary according to both race and ethnicity; collapsing racial/ethnic groups can alter means and distributions, generating faulty conclusions. Further research is warranted to explore relationships between dietary self-care and marital status, race, ethnicity, and physiological variables. ^

Molecular genetic analyses of extracellular signaling pathways during murine retinal development

Extracellular signaling pathways initiated by secreted proteins are important in the co-ordination of tissue interactions in multi-cellular organisms, particularly during embryonic development. These signaling cascades direct diverse cellular events, including proliferation, differentiation and migration, in both autocrine and paracrine modes. In adult animals, abnormal function of these proteins often results in degenerative and tumourigenic syndromes. In this study, I have focused on elucidating the role of Bone Morphogenetic Protein (Bmp) signal transduction during neuronal specification and differentiation in the vertebrate embryo, using the mouse retina as a model. Using tissue-specific conditional knock-out approaches, the consequences of genetic loss-of-function of this signaling pathway on retinal physiology were examined. Mutant mice lacking Bmp type I receptor function displayed a range of retinal phenotypes, each of which appeared to be regulated at a different threshold of Bmp receptor activity. Novel essential functions for Bmp signaling were uncovered for retinal neurogenesis, cell survival, and axonal pathfinding at the optic disc. Further, BmprIa and BmprIa exhibited genetic interactions suggestive of functional redundancy. To further characterize the underlying molecular bases for the pleiotropic effects of Bmp receptors, retina-specific loss-of-function mutants of the obligate Bmp-activated transcriptional mediator Smad4 were generated. A comparison of the retina-specific Smad4 mutant phenotypes with those of the Bmp receptor mutant retina revealed that only a subset of retinal phenotypes, namely optic disc axon pathfinding and axial patterning were common for both classes of mutant animals. Thus, these results suggest that, contrary to the classic scheme of Bmp signal transduction, Smad4-independent pathways may be operative downstream of the type I receptors. Indeed, such alternative intracellular signaling cascades may constitute a molecular basis for the multiple cellular responses elicited by Bmp signaling. Finally, I tested whether the potential Bmp pathway targets, the extracellular ligands Fgf9 and Fgf15, mediate essential cellular processes in the retina. The analyses of Fgf9 −/−; Fgf15−/− mutant mice posit a novel shared role for these genes in intra-retinal axon pathfinding. Collectively, these studies have elucidated part of the molecular machinery directing mammalian neuro-retinal development, and provided useful in vivo models to study visual function. ^

Relationship between Clostridium difficile toxin type and clinical features, severity and outcome in patients with C. difficile diarrhea (CDAD)

Background. Clostridium difficile infection is one of the major causes of antibiotic associated diarrhea and colitis in the United States. Currently, there is a dearth of literature on the risk factors and outcomes differences between the patients with infection due to the hypervirulent strain vs. the non-hypervirulent strains. The objective of this study was to determine the relationship between C. difficile toxin type and clinical features, severity and outcome in patients with C. difficile diarrhea. ^ Methods. The case group included 37 patients who had infections due to hypervirulent strain (tcdC deletion) and the control group included 55 patients with other toxin types (toxin A, B, binary toxin). A univariate analysis was performed followed by a multivariable logistic regression analysis to assess the differences between cases and controls. ^ Results. In the multivariate analyses, we found out that being a male was a protective factor for developing the infection due to the hypervirulent strain [OR 0.33; 95% CI 0.12-0.90]. Also, the hypervirulent group has worse clinical and economic outcomes, although the differences were small and nonsignificant. ^ Conclusions. There may likely be no predictive risk factor for acquiring infection due to the hypervirulent strain and the acquisition may be more linked to the infection control practices of the individual hospitals or location of patients. Hence, better infection control practices may prove helpful in decreasing the overall disease burden and thus improve patient outcomes. ^

The eukaryotic cellular stress response: Biochemical and genetic analyses in Saccharomyces cerevisiae

All cells must have the ability to deal with a variety of environmental stresses. Failure to correctly adapt to and/or protect against adverse stress conditions can lead to cell death. In humans, stress response defects have been linked to a number of neurodegenerative diseases and cancer, underscoring the importance of developing a fundamental understanding of the eukaryotic stress response.^ In an effort to characterize cellular response to high temperature stress, I identified and described one member of a novel gene family— RTR1. I show that the RTR1 gene and its protein product genetically and biochemically interact with core subunits of the RNA polymerase II enzyme. Appropriately, loss of RTR1 results in defective transcription from multiple promoters. These data provide evidence that Rtr1, which is essential under stress conditions, acts as a key regulator of transcription.^ In addition to transcriptional regulation, cells deal with many stressors by inducing molecular chaperones. Molecular chaperones are ubiquitous in all living cells and bind unfolded or damaged proteins and catalyze refolding or degradation. Hsp90 is a unique chaperone because it targets specific clients—typically signaling proteins—for maturation. While it has been shown that Sse1, the yeast Hsp110, is a critical regulator of the Hsp90 chaperone cycle, this work describes the molecular basis for that regulation. I show that Sse1 modulates Hsp90 function through regulation of Hsp70 nucleotide exchange. Further, Hsp110-type nucleotide exchange factors (NEFs) appear to have a specific role in modulating Hsp90 function in this manner. Finally, in addition to Hsp110, the eukaryotic cytosol contains two other types of Hsp70 NEF: Snl1 (BAG-domain protein) and Fes1 (HspBP1-like protein). I investigated the cellular roles of these NEFs to better understand the reason that eukaryotic cells contain three distinct protein families that perform the same biochemical function. I show that while cytsolic Hsp70 NEFs have some degree of functional overlap, they also exhibit striking divergence. Taken together, the work presented in this dissertation provides a more detailed understanding of the eukaryotic stress response. ^

Type A behavior and blood pressure during adolescence

In The Woodlands, Texas, 346 students in grades 9-12, age 14-18 participated in a screening examination for cardiovascular risk factors. The relationships between blood pressure with Type-A-behavior and its components were evaluated. Type-A-behavior was measured using the Hunter-Wolf Type-A-behavior scale.^ The following results refer to the current 24-item version of the Hunter-Wolf Type-A-behavior scale and subscales derived in the Bogalusa study which thereafter were applied to The Woodlands population.^ No significant differences in blood pressure were observed among children in the highest vs. lowest quintile of the Type-A-behavior score or subscales scores. The correlation coefficients of blood pressure with the Type-A-behavior and its components were small and non-significant in both boys and girls. Multiple regression analyses conducted by sex, showed that after adjustment for age, weight and height, the addition of the total Type-A-behavior score or subscale scores did not increase significantly the amount of the variability explained for any of the blood pressure components.^ These analyses were repeated with results from the original 17-item version of the Hunter-Wolf Type-A-behavior scale and subscales derived in Bogalusa. Similarly, no relationship was observed between the 17-item Type-A-behavior score or subscales scores with blood pressure levels in The Woodlands population.^ Finally, it was important to determine whether subscales derived within The Woodlands population would differ from those described in Bogalusa and would relate differently to blood pressure among students in The Woodlands. The corresponding analyses showed that the subscales derived from the two studies were different, but in fact neither set of subscales was importantly related with blood pressure in The Woodlands population.^ The results of this study are largely consistent with those obtained by Hunter and Wolf in Bogalusa, who among the white population found only the factor "Eagerness-Energy" to be associated with fourth phase diastolic blood pressure among girls. Even this relationship which they observed was weak and inconsistent across sex-race groups and blood pressure components. This study does not support even this positive finding. In conclusion, evidence indicates that blood pressure is not associated with Type-A-behavior or its components as measured by the Hunter-Wolf Type-A-behavior scale among white adolescents. ^

Genetic susceptibility, cigarette smoking, and wood dust exposure in lung cancer: Case control analyses

This case control study was conducted to assess the association between lung cancer risk, mutagen sensitivity (a marker of cancer susceptibility), and a putative lung carcinogen, wood dust exposure. There were 165 cases (98 African-Americans, 67 Mexican-Americans) with newly diagnosed, previously untreated lung cancer, and 239 controls, frequency-matched on age, sex, and ethnicity.^ Mutagen sensitivity ($\ge$1 break/cell) was associated with a statistically significant elevated risk for lung cancer (odds ratio (OR) = 4.1, 95% confidence limits (CL) = 2.3,7.2). Wood dust exposure was also a significant predictor of risk (OR = 2.8, 95% CL = 1.2,6.6) after controlling for smoking and mutagen sensitivity. When stratified by ethnicity, wood dust exposure was a significant risk factor for African-Americans (OR = 4.0, 95% CL = 1.4,11.5), but not for Mexican-Americans (OR = 1.5, 95% CL = 0.3,7.1). Stratified analysis suggested a greater than multiplicative interaction between wood dust exposure and both mutagen sensitivity and smoking.^ The cases had significantly more breaks on chromosomes 4 and 5 than the controls did with ORs of 4.9 (95% CL = 2.0, 11.7) and 3.9 (95% CL = 1.6, 9.3), respectively. Breaks at 4p14, 4q27, 4q31, 5q21-22, 5q31, and 5q33 were significantly more common in lung cancer patients than in controls. Lung cancer risk had a dose-response relationship with breaks on chromosomes 4 and 5. Cigarette smoking had a strong interaction with breaks on chromosomes 2, 4, and 5.^ In a molecular cytogenetic study, using chromosome painting and G-banding, we showed that: (1) the proportion of chromosome 5 abnormalities surviving as chromosome-type aberrations remained significantly higher in cells of lung cancer cases (14%) than in controls (5%) (P $<$ 0.001). However, no significant differences were detected in chromosome 4 abnormalities between cases and controls; (2) the proportion of chromosome 5q13-22 abnormalities was 5.3% in the cases and 0.7% in the controls (P $<$ 0.001). 5q13-22 regions represented 40% of all abnormalities on chromosome 5 in the cases and only 14% in the controls.^ This study suggests that mutagen sensitivity, wood dust exposure, and cigarette smoking were independent risk factors for lung cancer, and the susceptibility of particular chromosome loci to mutagenic damage may be a genetic marker for specific types of lung cancer. ^

RELATION OF TYPE OF REPORTED ILLNESSES TO PATTERN OF USE OF THE STUDENT HEALTH CENTER BY THE INTERNATIONAL STUDENTS AT TEXAS SOUTHERN UNIVERSITY, HOUSTON, TEXAS

The state of knowledge on the relation of stress factors, health problems and health service utilization among university students is limited. Special problems of stress exist for the international students due to their having to adjust to a new environment. It is this latter problem area that provides the focus for this study. Recognizing there are special stress factors affecting the international students, it is first necessary to see if the problems of cultural adaptation affect them to any greater degree than American students attending the same university.^ To make the comparison, the study identified a number of health problems of both American and international students and related their frequency to the use of the Student Health Center. The expectation was that there would be an association between the number of health problems and the number of life change events experienced by these students and between the number of health problems and stresses from social factors. It was also expected that the number of health problems would decline with the amount of social support.^ The population chosen were students newly enrolled in Texas Southern University, Houston, Texas in the Fall Semester of 1979. Two groups were selected at random: 126 international and 126 American students. The survey instrument was a self-administered questionnaire. The response rate was 90% (114) for the international and 94% (118) for the American students.^ Data analyses consisted of both descriptive and inferential statistics. Chi-squares and correlation coefficients were the statistics used in comparing the international students and the American students.^ There was a weak association between the number of health problems and the number of life change events, as reported by both the international and the American students. The study failed to show any statistically significant association between the number of stress from social factors and the number of health problems. It also failed to show an association between the number of health problems and the amount of social support. These findings applied to both the international and the American students.^ One unexpected finding was that certain health problems were reported by more American than international students. There were: cough, diarrhea, and trouble in sleeping. Another finding was that those students with health insurance had a higher level of utilization of the Health Center than those without health insurance. More international than American students utilized the Student Health Center.^ In comparing the women students, there was no statistical significant difference in their reported fertility related health problems.^ The investigator recommends that in follow-up studies, instead of grouping all international students together, that they be divided by major nationalities represented in the student body; that is, Iranians, Nigerians and others. ^

Correlation of blood type with thrombosis in pancreatic cancer patients

Objective: The study aimed to identify the risk factors involved in initiating thromboembolism (TE) in pancreatic cancer (PC) patients, with focus on ABO blood type. ^ Methods and Patients: There were 35.7% confirmed cases of TE and 64.3% cases remained free of TE (n=687). There were 12.7% only Pulmonary embolism (PE), 9% only Deep vein thrombosis (DVT), 53.5% only other sites, 3.3% combined PE and DVT, 8.6% combined PE and other sites, 9.8% combined DVT and other sites, and 3.3% all three combined cases. ^ Results: The risk factors for thrombosis identified by multivariate logistic regression were: history of previous anti-thrombotic treatment, tumor site in pancreatic body or tail, large tumor size, maximum glucose category more than 126 and 200 mg/dL. ^ The factors with worse overall survival by multivariate Cox regression and Kaplan Meier analyses were: locally advanced or metastatic stage, worsening performance status, high CA 19-9 levels, and HbA1C levels more than 6 %, at diagnosis. ^ There were 29.1% and 39.1% of the patients with thrombosis in the O and non-O blood type groups respectively. Both Non-O blood type (P=0.02) and the A, B and AB blood types (P= 0.007) were associated with thrombosis as compared to O type. The odds of thrombosis were nearly half in O blood type patients as compared to non-O blood type [OR-0.54 (95% C.I.- 0.37-0.79), P<0.001]. ^ Conclusion: A better understanding of the TE and PC relationship and involved risk factors may provide insights on tumor biology and patient response to prophylactic anticoagulation therapy.^