Browsing by Author "Phillips, Nicole R."
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Item An assessment of qPCR assays for DNA concentration and degradation(2019-05) Cropper, Emily R.; Coble, Michael D.; Warren, Joseph E.; Phillips, Nicole R.Forensically challenged samples are often composed of degraded, damaged, or low template mitochondrial DNA (mtDNA). A real-time quantitative polymerase chain reaction (qPCR) assay can help determine if there is sufficient quantity and robust quality of mtDNA to move forward with downstream sequencing and analysis. The fundamental issue with qPCR is that the nominal quantity of the DNA calibrated along the commercial standard used for quantification can vary depending on the supplier and lot numbers. The National Institute of Standards and Technology (NIST) has developed a commercially available human DNA standard, Standard Reference Material (SRM) 2372a, which consists of nuclear DNA (nDNA) and mtDNA data on three wellcharacterized human genomic DNA preparations. The SRM 2372a was used to compare three qPCR assays: a non-commercial triplex assay, for mtDNA quantification, and two commercial assays, Quantifiler Trio (QFTrio) for nDNA quantification, and NovaQUANT for nDNA quantification and determination of the mtDNA/nDNA ratio. Quantification of the SRM uniformly across these three qPCR assays allowed for the conclusion that a robust, reproducible, accurate, and efficient qPCR assay is dependent on (1) the quality and reliability of the DNA standard, (2) the specificity of the qPCR chemistry, and (3) sound primers and probes, to name a few. The findings indicate that commercially available qPCR assays do not necessarily perform as marketed and should be re-verified by a validated DNA SRM.Item Ancestry Informative Markers Tailored to Hispanic Populations(2020-05) Setser, Casandra H.; Cross, Deanna S.; Planz, John V.; Barber, Robert C.; Phillips, Nicole R.; Krishnamoorthy, Raghu R.Hispanic populations are highly heterogeneous despite being grouped together as a conglomerate population; this makes an accurate panel of ancestry informative markers (AIMs) especially important for human identification. In Chapter 2, the Genomic Origins and Admixture in Latinos (GOAL) dataset containing 494,886 SNPs was used for SNP ascertainment. Utilizing a country attributable variant of Wright's FST, 234 SNPs were selected for biogeographic ancestry (BGA) determination by tailoring each SNP to genetic differentiation of specific populations. Accuracy of BGA prediction was tested using multinomial logistic regression (MLR) and as few as 55 SNPs were robust to 90% for all populations studied. The panel of 234 SNPs was compressed by 65.8% to 80 SNPs by decreasing the influence of Honduras and the Dominican Republic SNPs with high country attributable mean FST values in favor of additional SNPs for Colombia, Cuba, and Puerto Rico; this balanced small panel size with classification accuracy. In Chapter 3, the Setser80 Hispanic AIMs panel was tested against the panels of 128 SNPs developed by the Seldin group and 55 SNPs developed by the Kidd group using STRUCTURE, PCA, a naive Bayesian classifier and MLR. In STRUCTURE, the Setser80 was able to distinguish Honduras, the Dominican Republic, and Colombia at K=4, where the Seldin and Kidd panels were optimized at K=3 and distinguished only Honduras and the Dominican Republic; similar results were obtained by PCA. The GOAL dataset was combined with the Admixed American super-population from the 1000 Genomes Project to test the panel on an expanded dataset of seven populations. Overall, the Setser80 had superior results to the Seldin and Kidd panels with 91.5% accuracy by naive Bayesian classifier and 93.2% by MLR. As an indication of its portability, the Setser80 had accuracies of >98% for Peru and >80% for Mexicans living in Los Angeles, which were not involved in SNP ascertainment. Given its accuracy and lack of overlap, the Setser80 may supplement existing panels for more granular Hispanic BGA determination. In Chapter 4, the application of allele frequencies to forensic genetics, genealogy, and clinical genetics are discussed as well as future directions and ethical considerations.Item Association between GDF5 single nucleotide polymorphism rs143383 and chronic lower back pain(2019-03-05) Phillips, Nicole R.; Aryal, Subhash; Licciardone, John C.; Tran, ApolloIntroduction. Low back pain presents a unique and ongoing challenge for patients and physicians. Of those who experience an episode of low back pain, 10% go on to develop persistent chronic low back pain (CLBP). However, the cause of this progression is not understood and it is unclear why the clinical manifestation of CLBP differs across individuals. There is a large body of evidence demonstrating the role of genetics as a risk factor CLBP. Growth factor differentiation factor 5 (GDF5) is a protein involved in the growth and development of bone and cartilage. A variant of GDF5, single-nucleotide polymorphism (SNP) rs143383 has been implicated with increased susceptibility and severity of musculoskeletal disorders such as osteoarthritis, rheumatoid arthritis, and ankylosing spondylitis. Considering that the cause of low back pain often involves musculoskeletal pathology, rs143383 may be implicated with symptomatology and the progression to persistent CLBP. Objective. This study seeks to determine whether the rs143383 SNP is associated with pain severity in CLBP. We hypothesize that subjects with the CC genotype experience higher levels of pain compared to the TT and CT genotypes. Methods. This project is an observational cohort study based on data retrieved from The Pain Registry for Epidemiological, Clinical, and Interventional Studies and Innovation (PRECISION). Subjects were divided into three groups, TT, CT, and CC. Average pain levels based on the Numerical Rating Scale (NRS) for low back pain were compared among the groups. Results. Using a general linear model, we found that the rs143383 SNP was significantly associated with NRS scores (P = 0.001). We also found that the CC genotype had a statistical higher mean NRS score than the CT (P = 0.0370) and TT (P = 0.0004) genotypes. However, when the data was adjusted for race, ethnicity, gender and age, no significance was found between rs143383 and NRS scores. Conclusion. Our findings indicate that there is no association between the GDF5 rs143383 polymorphism after adjusting for race and ethnicity. We were unable to complete a stratified analysis due to the distribution of participants in each strata. Larger studies should consider a stratified analysis to determine whether there is an association between rs143383 and CLBP within different ethnicities and racial groups.Item Association of ABCB1 (rs1045642) single nucleotide polymorphism and drug metabolism reserve index (DMRI) with pain intensity among adults with chronic low back pain(2019-03-05) Licciardone, John C.; Phillips, Nicole R.; Salman, JustinPurpose Low back pain is the leading cause of disability in the United States. It is often associated with long term opioid use, which may lead to opioid misuse and serious adverse events. Current clinical guidelines caution that opioids may provide only a small to moderate therapeutic effect, despite these potential risks. This study explores the use of pharmacogenetics in patients with chronic low back pain who are managed with opioids by investigating nucleotide variants in ABCB1,a gene implicated in drug bioavailability. Additionally, a three gene model using CYP2D6, CYP2C9, and CYP2C19, will be used to determine opioid drug metabolism on a quantitative scale. We aim to identify pharmacogenetics variants that may lead to safer and more effective treatment of chronic low back pain in patients being managed with opioids. Methods This study included 102 patients with low back pain within the Pain Registry for Epidemiological, Clinical, and Interventional Studies and Innovation (PRECISION). All patients in the study reported using opioids for their low back pain. DNA genotyping of biological samples from all patients was conducted on Illumnina iScan Global Screening Array. A common SNP locus (C3435T, rs1045642) in ABCB1was genotyped. Additionally, SNPs for CYP2D6, CYP2C9, and CYP2C19 were used to construct the drug metabolism reserve index (DMRI) for each patient, which was then stratified as Sub-functional (DMRI 6). Outcome measures included a numerical rating scale for low back pain intensity, the Roland-Morris Disability Questionnaire, and the PROMIS-SPADE cluster for quality of life. The Mann-Whitney test was used for statistical analysis in SPSS. Results Patients with the T allele at rs1045642, which inhibits ABCB1 protein function and increases drug bioavailability, reported significantly lower pain intensity within the functional DMRI group (p = 0.01). However, there was no significant difference in pain intensity with the sub-functional DMRI group (p = 0.186). Conclusion The results suggest that gene variants in ABCB1 may potentially affect pain relief in opioid users, and may be useful in guiding opioid prescribing for pain management. Longitudinal pharmacogenetic studies in larger cohorts are necessary to establish the utility of such gene variants in ABCB1 in guiding safer and more effective pain management in patients with low back pain.Item Association of Pain Sensitivity with Outcome Measures for Quality of Life, Functional Ability and Current Pain Intensity in Chronic Low Back Pain(2021-05) Doud, Ronnie L.; Sumien, Nathalie; Licciardone, John C.; Kearns, Cathleen; Phillips, Nicole R.Pain sensitivity, as measured by the self-reported Pain Sensitivity Questionnaire (PSQ), was investigated using data from the PRECISION Pain Research Registry. Outcome measures for quality of life, functional ability, and current pain intensity were found to be significantly associated with PSQ Total in participants reporting chronic low back pain, even when controlling for age, sex, race, ethnicity, smoking status, and body mass index. Higher reported pain sensitivity correlated with reported higher pain intensity, lower quality of life, and increased physical disability.Item Candidate gene analysis of 535 "pain genes" and associations with reported intensity of chronic low back pain(2022) Hurd, Christine A.; Phillips, Nicole R.; Lin, Emily; Broadbent, Dallen; Doederlein, Alexander R.; Dubakula, Vishnu; Licciardone, John C.Purpose: Numerous genome-wide association studies have been able to elucidate potential underlying genetic associations with clinical diagnoses. Chronic low back pain (CLBP) is a clinical presentation that has not yet been strongly associated with specific genetic markers. Several studies however have found genetic associations with other various pain disorders, such as the 535 genes identified by Ultsch et al. as "pain genes." Our group aims to find associations between previously identified pain-related genes and clinical reports of the intensity of low back pain using genetic and clinical data collected by the PRECISION Pain Research Registry. Methods: The PRECISION Pain Research Registry is a national registry that has collected demographic, clinical, and genetic information of patients with CLBP. Our analysis is querying associations between these identified "pain genes" and the intensity of low back pain reported by registry participants using a numerical rating scale (NRS). Methods: Participants in the PRECISION Pain Research Registry were genotyped via an Illumina iScan Array Scanner and Global Screening Array. The phenotype of CLBP will be measured by the NRS, which is an 11-point quantifier of pain intensity. The collected genotypes and phenotypic expression of pain will be compared via the Multi-marker Analysis of GenoMic Annotation (MAGMA), which enables candidate gene analysis of the 535 "pain genes" via congregation of single nucleotide polymorphisms (SNPs) and subsequent projection onto principal components in a matrix. Pain intensity will be evaluated as a function of genetic effects accounting for selected covariates-comorbid conditions with a documented relationship to CLBP, smoking status, and genetic ancestry plus residuals, with F-tests to determine the p-values of associations. Results: FN1 and STARD13 were found to be significantly associated with pain intensity in AA registry participants and VEGF-A was found to be significantly associated with widespread pain in NHW registry participants Conclusion: For treatment that is refractory to other strategies, targeted drugs for these protein products can be explored as treatments. These mentioned genes also have significant epigenetic regulation that could be explored in further studies.Item Candidate Gene Association Study of Low Back Pain Using SNP Derived Gene Expression Profiling: A PRECISION PAIN Research Registry Study(2019-03-05) Pathak, Gita; Aryal, Subhash; Phillips, Nicole R.; Licciardone, John C.; Bhatnagar, ShwetaCandidate Gene Association Study of Low Back Pain Using SNP Derived Gene Expression Profiling: A PRECISION PAIN Research Registry Study Shweta Bhatnagar, Gita Pathak, Subhash Aryal, Nicole Phillips, John Licciardone Abstract Objective: The Global Burden of Disease Study estimated that 632 million persons worldwide are affected by low back pain (LBP), making it the leading cause of disability worldwide. Furthering our understanding of genetic-based risk for LBP may allow for development of targeted gene therapy for pain which may help mitigate the healthcare and financial burden. Inflammatory genes have been implicated in pain disorders. Variability in how these genes are expressed may determine their association in low back pain. This study aims to predict the expression of candidate genes and their association with pain in participants of the PRECISION Pain Research Registry. Hypothesis: Elevated self-reported pain intensity and disability from LBP is associated with the higher expression of inflammatory genes. Methods: The DNA was collected, extracted, and genotyped using the Infinium® Global Screening Array (Illumina). Data were filtered based on standard quality control protocols (Anderson et al., 2010). Gene expression data of monocytes from the Multi-Ethnic Study for Atherosclerosis (MESA) was used for gene expression imputation using PrediXcan. Twenty-six candidate genes involved with inflammation and immune response processes (based on Gene Ontology Analysis) were analyzed. The imputed gene expression levels were transformed to dichotomized gene expression levels, over-expressed and under-expressed. The highly expressed gene levels were then tested for association with PRECISION participant outcomes data, including Roland-Morris Disability Score and a pain intensity score using SPSS. Results: Seven genes showed positive correlation between their predicted expression levels and scores on the Roland-Morris Questionnaire and the pain intensity scale for LBP: STAT-1, STAT-2, HLA-A, CD48, CD209, CLEC4G and SLAMF8. Also, as expected, many of these genes demonstrated co-expression patterns due to their common role in immune mediation. Conclusions: The results demonstrate a positive correlation between the increased expression of inflammatory genes and how the subjects perceived and reported LBP. Understanding the relationship between pain and variability in inflammatory genes could play a role in future precision medicine and pain management.Item Case-Case Genome-Wide Association Study of Age-Related Cancer and Alzheimer’s Disease(2017-03-14) Barber, Robert; Phillips, Nicole R.; Olmstead, KeeganBackground: Research over the past five years has strengthened in support of an inverse epidemiological correlation between Alzheimer’s disease (AD) and cancer--individuals with cancer are less likely to develop AD and those with AD have reduced cancer risk. Since cancer is characterized by uncontrolled cell division, and AD by neuronal death (and limited neuronal regeneration), this inverse relationship may point to dysregulation in some common underlying pathways. Here, we aim to investigate the genetic underpinnings of this unique relationship which have not been fully explored, using a unique case-case genome-wide association study (GWAS) design between an AD and cancer cohort. Hypothesis: We hypothesize that suggestive association signals will be observed when comparing the AD to cancer group, with the most interesting signals being those that are stronger when comparing cases-to-cases than when comparing cases-to-controls. Methods: Genome-wide SNP data for AD, Cancer, and Control groups were created using two publically available datasets: Breast Cancer (BrCa) and Prostate Cancer (PCa) Cohort Consortium and Alzheimer’s Disease Neuroimaging Initiative. Breast and prostate cancer were combined to form the Cancer group, which according to Cancer Research UK, are the most prevalent forms of adult and elderly cancers. All samples were typed with the Illumina Human610-Quad BeadChip. Rigorous data management and quality control measures were taken: group matching, updating map location, permutations test, sex check and filtering of low genotyping individuals and loci as well as loci with HWE issues. Three association analyses were performed: AD–Control, Cancer–Control, and AD–Cancer. Results: After matching for age, gender, and Caucasian ethnicity 492 individuals were included in the AD group (Avg age: 75 years, 37% female), 691 individuals in Cancer group (Avg age: 67.7 years, 37% female), and 1150 individuals in the combined Control group (avg age: 71 years, 37% female). Association analysis of the AD–Cancer study indicated one marker, rs2075650, as significant at p -8. Initial analysis also indicated possible clustering of significant SNPs on chromosomes 8 and 11. Conclusions: Case-case GWAS provides a novel means for identifying novel loci involved in the dichotomous relationship of risk of AD and risk of BrCa/PCa. These signals may point to critical genomic regions involved in age-related pathologies of cancer and AD.Item Cell-Free mtDNA Quantification in Alzheimer's Patients from the Mexican American Population(2020-05) House, Sara R.; Phillips, Nicole R.; Hodge, Lisa M.; Zascavage, Roxanne R.Abstract Background AD is a continuous problem in the 65+ population but it is especially challenging in the Hispanic population where not only is it more prevalent but more severe than Caucasian populations. This study explores the efficacy of using peripheral blood plasma as an alternative tissue for testing as well as the usefulness for future research assisting in identifying the population structure most at risk for developing AD based upon CF-mtDNA quantity results. Materials and Methods Samples tested included a total cohort (Mexican American and Caucasian) of 177 individuals (AD=45, MCI=74, NC=58). The Mexican American subset contained 92 individuals (AD=21, MCI=53, and NC=18). Peripheral blood plasma was collected from the TARCC biobank and quantified. CF-mtDNA was then tested for significance using correlation analyses, logistic and linear regression models. Results CF-mtDNA was significantly negatively correlated with education, age, sex, and hypertensive samples in the total and Mexican American populations. The greatest difference was expected to be in CF-mtDNA quantity from NC to AD samples. Instead, the most significant difference was between MCI and NC samples. As CF-mtDNA quantity increased, the MMSE and CDRSOB scores were less impaired. Conclusion In conclusion, CF-mtDNA is an easily accessible and easily tested molecular marker of diseases that are relevant to studies for cognitive decline. Although our findings were inconsistent with current literature, they bring to light the weight of confounding factors within limited sample studies. With the completion of the full sample set associated with this study, more power is needed to overcome these issues.Item Differences in race-specific outcome measures for chronic low back pain patients with relation to HTR2A variations(2021) Holden, Jeremy; Phillips, Nicole R.; Licciardone, John C.Purpose: The effects of many genes involved in the pathogenesis of chronic low back pain (CLBP) are not fully understood, especially concerning racial variation. This study aims to determine if variations of the serotonin receptor gene HTR2A, which has been implicated in psychological and pain disorders, correlate to differences in clinical outcome measures of patients with CLBP in the PRECISION Pain Research Registry. Methods: The base population includes 283 (68.4%) Caucasians and 131 (31.6%) African Americans who were genotyped for their haplotype in 2 haploblocks: A (rs6313;A/G, rs6311;T/C, rs1928040;A/G, rs9567746;A/G) and B (rs7997012;A/G, rs7330636;T/C). Race-specific Kruskal-Wallis analyses were used to determine differences in outcome measures when comparing haplotypes within haploblocks. Separate regression analyses looked at whether haplotypes that are overrepresented in one racial group versus the other had effects on the same outcomes. Results: There were differences in scores for the Roland-Morris Disability Questionnaire (RMDQ) (p=0.04), pain catastrophizing (PCS) (p=0.04), and pain self-efficacy (PSEQ) (p< 0.01) within haploblock A for African Americans. There were also differences in the RMDQ (p=0.02) and PSEQ (p< 0.01) scores within haploblock B for Caucasians. Regressions showed having at least one allele with the haplotype GC in haploblock B is associated with a lower numerical rating scale value for pain intensity after adjusting for additional variables (beta=-0.59; p=0.02). Conclusions: Haplotypes of HTR2A may have a relationship with the pain intensity, disability, and pain response of CLBP patients. Further studies would look at additional race-specific factors and their interplay with HTR2A.Item DNA repair polymorphisms and age-related diseases - Alzheimer’s and Cancer: Insights from SNP-set analysis and gene expression association(2018-03-14) Phillips, Nicole R.; Pathak, Gita A.Purpose: DNA repair response is a common thread for age-related diseases. Genomic stability is the result of an elaborate machinery consisting of damage response, repair, cell-cycle checkpoints, and apoptosis. A compromised DNA damage-repair response either due to time-dependent accumulation of damage or an individual’s reduced DNA repair capacity has been known to derail the genomic defenses, resulting in disease. Recent research findings and epidemiological studies speculate an inverse association between Alzheimer’s and cancer. Since impaired DNA repair is known to accelerate age-related disease, our goal is to evaluate DNA damage/repair genes and identify the role of DNA repair polymorphisms in Alzheimer’s, Breast and Prostate Cancer in individuals. Methods: The raw genotype and phenotype data were obtained via authorized access application for Alzheimer’s Disease Neuroimaging Initiative and Breast and Prostate Cancer Cohort Consortium; genotype data were generated using the Illumina Human Quad610™ Beadchip. Controls with positive family history were removed; all subjects used were [greater than] 50 years. Data were processed with in-house codes for QC, mapping SNPs to genes and extracting SNP sets based on 274 candidate genes. SNPs within each set were tested (permutation protocol, mperm=5000) and interpreted for biological relevance after correcting for multiple set-tests. Association analyses accounted for key covariates such as age and sex. Results with genomic inflation of more than 1.03 were adjusted using first three eigenvectors as covariates. Gene expression was imputed for candidate genes. Analyses were performed in Plink(v1.9), EIGENSOFT-6.4, Rstudio 3.4, Bioconductor 3.6, VEGA2, Python 3, PrediXcan and MAGMA. Results: After two-level QC filtering, the datasets – ADNI, Breast and Prostate cancer – had 677, 578 and 3857 individuals, respectively. Gene-sets of ~167 genes were created for each dataset. Preliminary results point to cancer-specific variants in key DNA repair genes, some of which have not previously been reported. Structured sets of DNA repair pathways and gene expression imputation are in the analysis phase. Conclusion: This study investigates DNA repair genes in both cancer and Alzheimer’s using SNP-set analysis to improve detection of association that sometimes get lost in whole-genome associations. Our results provide a detailed overview of various DNA repair genes and their association with complex phenotypes of age-associated diseases.Item Effects of a Synthetic Amino Acid Diet: Insights from the Guy Microbiome, Inflammation, and Behavior(2021-05) Mancilla, Viviana J.; Allen, Michael S.; Jones, Harlan P.; Phillips, Nicole R.; Planz, John V.; Ellis, DorettePhenylketonuria (PKU) is an inborn error of phenylalanine metabolism primarily treated through a phenylalanine-restrictive diet and frequently supplemented with an amino acid formula to maintain proper nutrition. PKU patients often report high levels of anxiety along with symptoms of gastrointestinal distress (i.e., chronic diarrhea, constipation, cramps); symptoms previously associated with gut microbiome dysbiosis. Little is known of the effects of these dietary interventions on the gut microbiome of PKU patients, particularly in adults. The gut microbiome is a collection of microbes residing primarily in the large intestine. The colon is a major production site for short chain fatty acids (SCFAs) through anaerobic fermentation by commensal bacteria. SCFAs provide a source of energy for the colonocytes, as well as provide anti-inflammatory benefits. The production of SCFA appears to be dependent on the availability of soluble fibers and members of the gut microbiota capable of fermentation. We characterized the gut microbiome of adults with PKU for the first time and identified signs of dysbiosis. We then focused on the synthetic, low fiber, nature of the amino acid diet in a murine model. In this interdisciplinary study, we monitored the effect of a consuming synthetic diet on the composition of the murine gut microbiome over the course of 13 weeks, beginning at weaning. At the conclusion of the feeding period, mice we observed for anxiolytic behavior, locomotion, and cognition. We also searched for markers of inflammation through colon shrinkage, changes in cytokine levels within several tissues, and determined the concentration of SCFAs in the colon at the conclusion of the feeding period. The gut microbiome of mice fed the synthetic diet experienced significant deviation from the control group which affected relative abundance of beneficial bacteria. Mice on the synthetic diet were found to have shorter colons, lower concentration of SCFAs in the colon, and demonstrated elevated exploratory behavior.Item Establishing an STR allele frequency database for the Paraguayan population(2018-05) Giuffrida, Stephanie M.; Planz, John V.; Warren, Joseph E.; Phillips, Nicole R.; Cunningham, J. ThomasThis project determined whether the loci included in the Athos PCR Directa para Identificacao Humana Amplification and AmpFlSTR[R] Identifiler[R] PCR Amplification Kits are suitable for establishment of an STR allele frequency database representative of the Paraguayan population. Allele frequencies for the 21 STR loci included in the Athos PCR Directa para Identificacao Humana Amplification Kit were calculated for 200 individuals in the Paraguayan population. Allele frequencies for the 15 STR loci included in the AmpFlSTR[R] Identifiler[R] PCR Amplification Kit were also calculated for 300 individuals in the Paraguayan population. Performance of these loci was determined by testing the population for Hardy-Weinberg Equilibrium, testing for Linkage Disequilibrium between the loci in the kits, and calculating Power of Discrimination, Power of Exclusion, and Mean Power of Exclusion. Comparability between the common loci included in the two PCR kits was also tested. Results of these tests determined that the Athos PCR Directa para Identificacao Humana Amplification and AmpFlSTR[R] Identifiler[R] PCR Amplification Kits are both suitable for use in an STR allele frequency database. Additionally, the common loci contained in the two PCR kits are comparable, and the populations can be combined to create one database.Item Examining SLC6A4 Variations in the PRECISION Pain Research Registry(2019-03-05) Phillips, Nicole R.; Aryal, Subhash; Licciardone, John C.; Lopez, JonathanPurpose: Chronic low back pain is the leading cause of disability globally and has been linked to comorbidities such as depression. Common pathways involving serotonin and norepinephrine may play a role in both pain and mood disorders. The SLC6A4 gene, encoding a serotonin reuptake transporter, has been heavily studied with regard to depression. Polymorphisms within the gene are thought to influence the expression of the transporter, thereby modulating serotonin transmission. More recently, this gene has been studied in the context of chronic pain. This study seeks to further our understanding of chronic pain with respect to depression and other outcome measures in participants with chronic low back pain to aid in the development of better treatments. Methods: Participants with chronic low back pain in the PRECISION Pain Research Registry provided DNA samples that were genotyped on the Infinium Global Screening Array (Illumina). Long and short length polymorphisms of the SLC6A4 gene were predicted using an eight single nucleotide polymorphism (SNP) machine learning model. Of the eight, one SNP was collected from the array, and the other seven were imputed. Additionally, the rs25531A [greater than] G SNP was imputed. Using the length of the polymorphism and the rs25531 SNP, subjects were divided into high, intermediate and low expression groups. Participants also reported clinical status measures such as low back pain intensity, back-specific functioning, PROMIS quality of life, levels of pain self-efficacy, and pain catastrophizing. Results: There was no significant association between self-reported depression and expression levels (high, intermediate, low) of SLC6A4. No correlation was found between PROMIS depression scores and SLC6A4 expression. Analyses for depression values were conducted using logistic linear regression and non-parametric ANOVA respectively. There were no observed correlations between transporter expression level and the outcome variables of back-related disability, pain, pain catastrophizing, or pain self-efficacy. Disability was analyzed using ANOVA. Pain, pain catastrophizing, and pain self-efficacy were investigated using non-parametric ANOVA analyses. Conclusion: No correlations were found between serotonin transporter expression level and depression or other outcome variables. Similar previous studies investigating SLC6A4 used homogenous populations. We recommend conducting a larger study that takes into account race.Item Expert Systems for High Throughput Analysis of Single Source Samples: A Comparison of GeneMarker® HID v1.71 and GeneMapper® ID v3.2 and Validation of GeneMapper® ID v3.2(2009-05-01) Phillips, Nicole R.; Planz, John V.The NIJ Convicted Offender DNA Backlog Reduction Program has helped laboratories in the U.S. to enhance throughput of single source sample processing technologies and methods with the adoption of robotics, single amplification kits and multicapillary instruments. With these advances, the bottleneck in forensic laboratories today is data analysis. Commercially available expert systems offer automated sizing and analysis, expediting the process of DNA data analysis. The University of North Texas Center for Human Identification Research & Development Laboratory has been contracted for several population database construction projects, requiring a high throughput approach for genetic processing of the single source samples. Summarized in this report are the comparison of two expert systems and the internal validation of a GeneMapper® ID v3.2 (Applied Biosystems, Foster City, CA) as an expert system. GeneMarker® HID v1.71 (SoftGenetics, State College, PA) and GeneMapper® ID v3.2 are the two expert systems chosen for the initial evaluation. The features, user interface, and performance of each software program were compared. GeneMarker HID and GeneMapper ID performed similarly, making accurate allele calls and appropriately directing the analyst's attention to data that do not meet defined thresholds. The decision was made to internally validate GeneMapper ID as an expert system for STR data analysis of single source samples. The expert system was successfully optimized for the analysis of samples amplified with Identifiler® and Yfiler® PCR Amplification Kits (Applied Biosystems)and analyzed on the 3130xl Genetic Analyzer (Applied Biosystems).Item Expression and Function of Ligands for Natural Killer Cell Receptors on Triple-negative Breast Cancer Cells(2018-08) Marrufo, Armando M.; Mathew, Porunelloor A.; Mathew, Stephen O.; Jones, Harlan P.; Phillips, Nicole R.Triple-negative breast cancer (TNBC) accounts for 20 percent of all breast cancer cases and is known to be the most invasive form of breast cancer. TNBC's absence of estrogen, progesterone, and human epidermal growth factor-2 receptors makes utilizing hormonal treatments ineffective in suppressing tumor growth. TNBC is associated with poorer prognosis and higher incidences of relapse. Therefore, natural killer cell-mediated immunotherapy shows potential as a treatment option for TNBC. Natural killer cells (NK) are innate lymphoid cells that serves its role in the immune system to eradicate infected and tumor cells. NK cell function is regulated through its receptors interacting with activating and inhibitory ligands on target cells. Lectin-like Transcript-1 (LLT1, CLEC2D) is a ligand that interacts with NKRP1A (CD161) and inhibits NK cell activation. Proliferating Cell Nuclear Antigen (PCNA) is a ligand that interacts with NKp44 and inhibits NK cell activation. We have identified the expression and function of LLT1 and PCNA on TNBC cell lines by flow cytometry, western blot, immunofluorescent microscopy, and chromium-release assay. Our results have demonstrated a higher expression of LLT1 and PCNA on TNBCs than non-tumorigenic breast cell line MCF10A. We have shown that blocking LLT1 interaction with NKRP1A with antibodies and gene knockdown of LLT1, respectively, on TNBCs have increased lysis of TNBCs by primary NK cells. We have also shown that blocking PCNA interaction with NKp44 with antibodies have enhanced killing of TNBCs by NK cells. LLT1 and PCNA expressed on TNBCs sends an inhibitory signal to the NK cell thus serving its role for TNBCs to evade immunosurveillance. Blocking LLT1-NKRP1A or PCNA-NKp44 with antibodies enhances lysis by NK cells and may open a novel immunotherapeutic strategy for patients diagnosed with TNBC.Item Genetic characterization of comorbidity patterns in aging associated diseases using integrative genomics(2019-08) Pathak, Gita A.; Phillips, Nicole R.; Planz, John V.; Barber, Robert C.; Zhou, Zhengyang; Gryczynski, IgnacyThe aging population in the US continues to grow at an exponential rate estimated to reach more than 90 million by 2060. The coexistence of two or more diseases (comorbidity) is prevalent in ages 65 years and above, and the number of comorbidities increases with age. The genetic factors underlying presence and absence of comorbidities is a severely understudied research domain. Alzheimer's disease (AD) is a type of dementia affecting 5.5 million people with an average age of diagnosis at 70 years. Hypertension is a coexisting condition in 60% AD individuals, also known as direct comorbidity. On the other hand, cancer is reported to be inversely comorbid with AD; individuals with cancer history have been reported to have lower risk of AD and vice versa. Furthermore, individuals with cancer history are diagnosed with long term side effects of radiation therapy — radiotoxicity. Twin-based studies have reported that certain gene variants are associated with radiotoxicity phenotypes with a heritability of 66%. This study proposes to investigate genetic factors associated with the direct and inverse comorbidity of AD with hypertension and cancer, and proctitis — a radiotoxicity phenotype observed in survivors of prostate cancer. The study aims to integrate gene variants, derived-gene expression and copy number variation (CNV), followed by functional and pathway-based prioritization of observed findings. We used genome-wide and cerebral spinal fluid profile to investigate presence of hypertension with AD to evaluate individual-level differences, followed by targeted investigation of neighboring gene expression profiles of identified variants. We found several novel genes associated with AD-hypertension comorbidity. The investigation between AD and cancer identified regions in chromosomes 4, 5 and 19 that are targeted by miRNA-17 family along with other miRNAs reported to be inversely expressed and play opposite role in pathogenicity of both diseases. The SNP-derived transcriptomic profile between AD and cancer highlighted involvement of sirtuin signaling. The findings together indicate involvement of mitochondrial and metabolic dysregulation which possibly contribute in differences of the epithelial-mesenchymal-transition. The SNP-derived expression and CNV association with proctitis highlighted genes involved in DNA-repair and mitochondrial ROS damage pathways.Item Genetic Influences on Opioid Use in Low Back Pain: OPRM1 rs1799971(2019-03-05) Phillips, Nicole R.; Licciardone, John C.; Romanowski, BenjaminPurpose: The OPRM1 rs1799971 single nucleotide polymorphism (SNP) has been studied for its influence on drug abuse and opioid use. Opioids have a questionable long-term risk-benefit profile in chronic low back pain, and a genetic predisposition to taking higher doses could place a patient at higher risk of complications related to opioid use. Studies have demonstrated higher dosages of self-administered opioids in an acute post-surgical setting in subjects with the rs1799971 AG polymorphism. We hypothesize that subjects with the rs1799971 AG polymorphism will use higher doses of opioids than subjects with other rs1799971 polymorphisms in the setting of chronic low back pain. Methods: This study was conducted using data from the PRECISION Pain Research Registry at UNTHSC. A saliva sample from each subject was obtained to determine genotypes, including the OPRM1 rs1799971 SNP. Additionally, numerical ratings of low back pain intensity and opioid use in morphine milligram equivalents (MMEs) were measured. The MMEs were computed in accord with CDC guidelines, which further indicate that opioid doses greater than 50 MMEs per day double the risk of opioid complications as compared with doses under 20 MMEs per day. We analyzed pain intensity, daily MMEs, and the proportion of subjects taking doses in excess of 50 MMEs per day based on allele status at SNP rs1799971. Results: Of 351 subjects with subacute or chronic low back pain, 279 were AA and 72 were AG. There was no significant difference in MMEs between rs1799971 AA subjects (x̅=5.90, SD=17.03) and AG subjects (x̅=9.53, SD=22.85). AG subjects had statistically significant lower pain intensity (x̅=5.2777 SD=1.74) compared to AA subjects (x̅=5.985, SD=1.9489 Mann-Whitney U=7897.5 p=0.005). rs1799971 AG subjects were more likely to be taking opioid doses greater than 50 MMEs per day than AA subjects (OR=3.40 95% CI:1.01-11.46 p=0.04). Five and 6 subjects with AG and AA, respectively, were taking doses greater than 50 MMEs per day (OR, 3.40; 95% CI, 1.01-11.46; Fishers exact p=0.11) Conclusion: There was no significant difference in mean MMEs among rs1799971 AG and AA subjects. However, AG subjects were marginally more likely than AA subjects to be taking doses greater than 50 MMEs per day. This SNP could potentially place rs1799971 AG patients at higher risk of complications relating to higher opioid doses, indicating a less favorable risk-benefit profile for long-term opioid therapy.Item Genetically-regulated transcriptomics & copy number variation of proctitis points to altered mitochondrial and DNA repair mechanisms in individuals of European ancestry(BioMed Central Ltd., 2020-10-02) Pathak, Gita A.; Polimanti, Renato; Silzer, Talisa K.; Wendt, Frank R.; Chakraborty, Ranajit; Phillips, Nicole R.BACKGROUND: Proctitis is an inflammation of the rectum and may be induced by radiation treatment for cancer. The genetic heritability of developing radiotoxicity and prior role of genetic variants as being associated with side-effects of radiotherapy necessitates further investigation for underlying molecular mechanisms. In this study, we investigated gene expression regulated by genetic variants, and copy number variation in prostate cancer survivors with radiotoxicity. METHODS: We investigated proctitis as a radiotoxic endpoint in prostate cancer patients who received radiotherapy (n = 222). We analyzed the copy number variation and genetically regulated gene expression profiles of whole-blood and prostate tissue associated with proctitis. The SNP and copy number data were genotyped on Affymetrix(R) Genome-wide Human SNP Array 6.0. Following QC measures, the genotypes were used to obtain gene expression by leveraging GTEx, a reference dataset for gene expression association based on genotype and RNA-seq information for prostate (n = 132) and whole-blood tissue (n = 369). RESULTS: In prostate tissue, 62 genes were significantly associated with proctitis, and 98 genes in whole-blood tissue. Six genes - CABLES2, ATP6AP1L, IFIT5, ATRIP, TELO2, and PARD6G were common to both tissues. The copy number analysis identified seven regions associated with proctitis, one of which (ALG1L2) was also associated with proctitis based on transcriptomic profiles in the whole-blood tissue. The genes identified via transcriptomics and copy number variation association were further investigated for enriched pathways and gene ontology. Some of the enriched processes were DNA repair, mitochondrial apoptosis regulation, cell-to-cell signaling interaction processes for renal and urological system, and organismal injury. CONCLUSIONS: We report gene expression changes based on genetic polymorphisms. Integrating gene-network information identified these genes to relate to canonical DNA repair genes and processes. This investigation highlights genes involved in DNA repair processes and mitochondrial malfunction possibly via inflammation. Therefore, it is suggested that larger studies will provide more power to infer the extent of underlying genetic contribution for an individual's susceptibility to developing radiotoxicity.Item Genome-wide study highlights novel genes associated with Alzheimer’s-Hypertension comorbidity showing utility over CSF biomarkers(2019-03-05) Phillips, Nicole R.; Pathak, Gita A.Purpose: The aging population (65 and older) is heavily burdened with comorbidities. As the incidence of Alzheimer’s continues to rise in the aging demographic, understanding underlying causes of prevalent comorbid patterns in Alzheimer’s (AD) is crucial for early diagnosis and treatment. According to the 2011 Alzheimer’s association report, hypertension is the most prevalent comorbid condition affecting 60% of the Alzheimer’s population. However, there are no known biomarkers or risk scores associated with the AD-hypertension comorbidity; additionally, the genetic underpinnings of hypertension as a comorbidity to AD remains understudied. Hypothesis: We hypothesize that genetic variants underlie comorbidity patterns of Alzheimer’s disease and hypertension, which are distinct from genetic risk factors of Alzheimer’s disease alone. Methods: Leveraging the data from the Alzheimer’s Disease Neuroimaging Initiative (ADNI), we conducted comorbidity analyses comparing healthy cohort (controls) vs Alz+/Hyp- vs Alz+/Hyp+. We compared CSF biomarkers – amyloid β, tau and p-tau in three cohorts using one-way ANOVA. We, then evaluated genome wide profiles in three groups using 535,762 SNP markers in 677 individuals (after QC). SNPs were clumped into genes based on position ± 50kb and p-value, followed by mining their role in gene-expression pathways. Results: The CSF biomarkers were not significantly different in the disease groups, indicating that these biomarkers are not able to discriminate between comorbid AD-hypertension pathology. When comparing the control with Alzheimer’s individuals, genome-wide study identifies known - TOMM40 and novel genes -PML and KMO which are known to have role in multiple CNS disorders. Interestingly, when comparing controls vs Alz+Hyp+, we observe several genes in the chromosome 16 region, including SLC9A3R2 - a known hypertension gene. This gene-cluster was also found to be co-expressed via other intermediate genes, underscoring their involvement in mitochondrial pathways. Conclusion: While the CSF biomarkers- amyloid β, tau and p-tau are known for their diagnostic contribution in vascular dementia, their profile is unaltered in Alzheimer’s-hypertension comorbidity, requiring investigation of other possible pathogenic causes. This study replicates genes known for Alzheimer’s, along with identifying possible risk loci to the developing Alzheimer’s and hypertension, thus showing potential utility over CSF profiles.
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