Protein Structure-Guided Approaches to Identify Functional Mutations in Cancer

Distinguishing driver mutations from passenger mutations within tumor cells continues to be a major challenge in cancer genomics. Many computational tools have been developed to address this challenge; however, they rely heavily on primary protein sequence context and frequency/mutation rate. Rare driver mutations not found in many cancer patients may be missed with these traditional approaches. Additionally, the structural context of mutations on tertiary/quaternary protein structures is not taken into account and may play a more prominent role in determining phenotype and function. This dissertation first presents a novel computational tool called HotSpot3D, which identifies regions of protein structures that are enriched in proximal mutations from cancer patients and identifies clusters of mutations within a single protein as well as along the interface of protein-protein complexes. This tool gives insight to potential rare driver mutations that may cluster closely to known hotspot driver mutations as well as critical regions of proteins specific to certain cancer types. A small subset of predictions from this tool are validated using high throughput phosphorylation data and in vitro cell-based assay to support its biological utility. We then shift to studying the druggability of mutations and apply HotSpot3D to identify potential druggable mutations that cluster with known sensitive actionable mutations. We also demonstrate how utilizing integrative omics approaches better enables precision oncology; Combining multiple data types such as genomic mutations or mRNA/protein expression outliers as biomarkers of druggability can expand the druggable cohort, better inform treatment response, and nominate novel combinatorial therapies for clinical trials. Lastly, we improve driver predictions of HotSpot3D by creating a supervised learning approach that integrates additional biological features related to structural context beyond just positional clustering. Overall, this dissertation provides a suite of computational methods to explore mutations in the context of protein structure and their potential implications in oncogenesis

Correlation of maternal age and combined assessments on risk of chromosomal anomaly during prenatal screening

Background: Prenatal detection of genetic abnormalities is one of the biggest challenges of current fetal medicine. Prenatal screening for chromosomal abnormalities can be done using biochemical tests. The screening is a risk estimation test and not a diagnostic test. Methods: Statistical data treatment had been performed on a sample of 362 pregnant women for prenatal screening. This was a retrospective data analysis study undertaken at the National Reference Laboratory, Redcliffe Labs. Results: Nine (2.48%) women out of 362 were screen positive for chromosomopathy. The point biserial correlation between variables (Free β-hCG - Free Beta Human Chorionic Gonadotrophin, PAPP-A- pregnancy associated plasma protein-A and NT-(nuchal translucency) amongst patients with positive and negative screen test was statistically significant. There was a positive correlation between positive screen for chromosomopathy and hCG, MoM, NT MoM whereas a negative correlation between them and PAPP-A. This study indicates that higher values of hCG and lower values of PAPP-A MoM as seen in the positive screen patients is associated with a significant risk of chromosomopathy. A positive correlation between age and screen positive cases was seen. The McNemar’s test indicated a significant reduction in screen positive cases when biomarkers were added to screen for Trisomy 21 in women aged >35 years (n=86). 81 women eventually screened negative. Conclusions: The analyses stresses on the importance of using state-of-the-art, prenatal noninvasive screening software to help provide a predictive outcome, individualized for that pregnant woman.

Establishment of age-specific reference intervals for AMH in Indian women and enhancing its use as a diagnostic marker in PCOS

Background: Anti Mullerian hormone (AMH) level is a reliable marker of ovarian reserve. It is known to be influenced by factors like age, ethnicity, and ovarian pathology. Establishment of age-specific reference intervals for AMH, characteristic of different nationalities, is therefore of utmost importance. Serum AMH is known to be elevated in women with polycystic ovarian syndrome (PCOS). It is desirable to determine a population-specific cut-off of AMH, for it to be used as a diagnostic marker for PCOS. Methods: Serum AMH, luteinizing hormone (LH), follicle-stimulating hormone (FSH), Estradiol, Progesterone and Testosterone assays were analyzed in 1978 Indian women, in the age range of 12–50 years. Age-specific reference intervals for AMH were derived for the study population. The cohort of study subjects were then divided into two groups, based on AMH values and clinical history: Control group, and patients with PCOS. The cut-off value of AMH in the study population, corresponding to the diagnosis of PCOS, was also established.   Results: Upper 95th percentile limits of reference intervals for the 18-25 26–30, 31–35, and 36–40, 41-45 and >45 age groups were 9.69, 7.60, 6.50, 6.1, 4.80 and 4.5 ng/ml respectively. In the PCOS group the 5th percentile value was 7.80 ng/ml and the upper 95th percentile was 21.81 ng/ml. The median percentile in PCOS group was 10.40 ng/ml. ROC analysis was done to obtain optimal cutoff values for each age group with better discriminative power than the reference limits. The best cut-off point of AMH value for PCOS in our study population was 7.51ng/ml. The sensitivity and specificity were 99.4% and 95.5%, respectively. The calculated area under the Receiver operating characteristic (ROC) curve was 0.988 (95% CI: 0.984-0.991, P <0.001). AMH, LH, and LH/FSH ratio was significantly higher in the PCOS group than in the control group (p < 0.001 for all comparisons). LH/FSH ratio was more than 2 in the PCOS group compared to controls. Serum Testosterone was significantly higher in PCOS. Conclusions: The study aids to establish a biological reference interval for AMH, specific for different age groups in Indian women. 7.51ng/ml has been derived as a diagnostic cut-off of AMH for PCOS in our study population. The establishment of age-specific reference intervals, and syndrome-specific cut-offs in the Indian population will help overcome the influence of variables and broaden the use of AMH in women’s health

HIV Interventions to Reduce HIV/AIDS Stigma: A Systematic Review

We reviewed the literature to determine the effectiveness of HIV-related interventions in reducing HIV/AIDS stigma. Studies selected had randomized controlled trial (RCT), pretest–posttest with a non-randomized control group, or pretest–posttest one group study designs in which HIV-related interventions were being evaluated, and in which HIV/AIDS stigma was one of the outcomes being measured. A checklist was used to extract data from accepted studies, assess their internal validity, and overall quality. Data were extracted from 19 studies, and 14 of these studies demonstrated effectiveness in reducing HIV/ AIDS stigma. Only 2 of these 14 effective studies were considered good studies, based on quality, the extent to which the intervention focused on reducing HIV/AIDS stigma, and the statistics reported to demonstrate effectiveness. Future studies to reduce HIV/AIDS stigma could improve by designing interventions that pay greater attention to internal validity, use validated HIV/AIDS stigma instruments, and achieve both statistical and public health significance

How Researchers Define Vulnerable Populations in HIV/AIDS Clinical Trials

In this study, we interviewed researchers, asking them to define vulnerable populations in HIV/AIDS clinical trials, and provide feedback on the federal regulations for three vulnerable populations. Interview data informed a conceptual framework, and were content analyzed to identify acceptability or disagreement with the regulations. Beginning with several characteristics of vulnerable enrollees identified by researchers, the conceptual framework illustrates possible scenarios of how enrollees could be considered vulnerable in clinical research. Content analysis identified barriers affecting HIV/AIDS researchers’ ability to conduct clinical trials with pregnant women, prisoners, and children, for which the regulations specify additional protections. This study challenges current thinking about federal regulations’ group-based approach to defining vulnerable populations

Pilot study demonstrating effectiveness of targeted education to improve informed consent understanding in AIDS clinical trials

Assessing and improving informed consent understanding is equally important as obtaining consent from participants in clinical trial research, but developing interventions to target gaps in participants’ informed consent understanding remains a challenge. We used a randomized controlled study design to pilot test an educational intervention to improve actual informed consent understanding of new enrollees in the Adult AIDS Clinical Trial Group (AACTG). Questionnaires were administered to 24 enrollees to assess their baseline understanding on eight elements of informed consent associated with AIDS clinical trials. Enrollees who scored 18/21(85%) or less were randomly assigned to in-person, targeted education (intervention) or delayed education (control). Two follow-up assessments were administered. Repeated measures ANOVA was performed to determine intervention effectiveness in improving actual informed consent understanding over time. Actual understanding improved at the immediate post-intervention time point with a significant score difference of 2.5 when comparing the intervention and delayed groups. In addition, there was a significant score difference of 3.2 when comparing baseline to 3-month follow-up for the two groups, suggesting a statistically significant intervention effect to improve actual understanding of the basic elements of informed consent. The findings demonstrated that one-time targeted education can improve actual informed consent understanding one week after the intervention, but retention of these concepts may require periodic monitoring to ensure comprehension throughout the course of a clinical trial