Browsing by Author "Phillips, Nicole"
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Item Absolute Quantification of Mitochondrial DNA in Peripheral Blood from Women with Preeclampsia(2020) Silzer, Talisa; Phillips, Nicole; Goulopoulou, Styliani; Reid, Danielle; Sun, Jie; Scroggins, Sabrina; Santillan, Mark; Santillan, Donna; Cushen, SpencerIntroduction Mitochondrial DNA (mtDNA) in maternal blood has been proposed as a potential predictor of preeclampsia (PE). The objective of this study was to use an absolute PCR (abPCR) quantification protocol to determine concentrations of mtDNA in maternal plasma and peripheral blood mononuclear cells (PBMC) from normal and PE pregnancies. Methods Blood samples were collected from pregnant women with uncomplicated pregnancies and pregnancies with PE (University of Iowa IRB#200910784). abPCR quantification of mtDNA and nDNA was performed on DNA extracts from plasma (in the presence or absence of lysis buffer) and PBMCs using TaqMan(TM) probes and chemistry. Results When plasma DNA was extracted using lysis buffer, mtDNA concentrations were lower in women with PE than in controls (Control: 4.83 ± 1.09 vs. PE: 1.72 ± 0.38 pg/uL, n=19, P=0.017), while concentrations of nDNA did not differ (P=0.39). Without lysis buffer, plasma mtDNA remained lower in women with PE compared to controls (Control: 0.0106 ± 0.0019 vs PE: 0.0019 ± 0.0003 pg/uL, n=16-20, P< 0.0001). There were no group differences in PBMC mtDNA (P=0.66) and nDNA (P=0.13) concentrations. Conclusion mtDNA concentrations were lower in plasma of pregnant women with PE compared to controls. A significant amount of mtDNA was membrane bound as indicated by a 480-fold greater concentration in DNA isolated from plasma with lysis buffer vs. without. Use of this improved method of quantification of mtDNA in multiple blood fractions may allow for its development as a biomarker to detect PE prior to the onset of organ damage.Item Alzheimer's Disease Risk Allele Frequencies Differ Based on Ethnicity in HABLE Cohort(2022) Housini, Mohammad; Rao, Sumedha; Phillips, Nicole; O'Bryant, Sid; Barber, Robert C.Purpose: Alzheimer's Disease (AD) or other related dementias remain a significant burden on our aging population. Here we evaluate the top 10 AD risk alleles previously reported by Kunkle et al. (2018) in Mexican Americans and non-Hispanic whites enrolled in the Healthy Aging Brain in Latino Elders Study (HABLE) cohort to see if allele frequencies vary based on ethnicity. Methods: DNA was extracted from buffy coat samples (n = 1635) on the Hamilton robotic system with the Mag-Bind Blood & Tissue DNA HDQ 96 Kit. Genotyping was performed per manufacturer's protocol using the Illumina Infinium Global Screening Array (GSA) and analyzed with Genome Studio 2.0. Samples with call rates less than 98% were repeated or excluded. Allele and genotype frequencies were calculated using standard statistics by compiling the top ten AD risk alleles from Kunkle et al. (2018) and measuring their frequencies in the HABLE cohort. Results: Our data suggest varying degrees of allele and genotype frequencies among the top 10 risk conferring SNPs between Mexican Americans and Non-Hispanic Whites. In particular, we show some instances (BIN1, PTK2B) where the heterozygotes are in higher frequency than homozygotes. 8 of our evaluated SNPs show a difference greater than 5% between the two ethnicities. Conclusion: It may be beneficial to further study the top AD risk alleles among different ethnicities to determine if there are variable frequencies in those populations. We plan to expand and continue this work in other ethnicities and further elaborate on these differences to promote ethnicity targeted diagnostics and help reduce health disparities in medicine and science.Item Analysis of 436,390 genetic variants in 9,765 elderly individuals implicates TOMM40, MARK4, CLPTM1 and VDAC1/FSTL4 in the inverse relationship between Alzheimer's and cancer(2019-03-05) Zhou, Zhengyang; Phillips, Nicole; Pathak, Gita A.Purpose A rapidly aging demographic, aged 65 and older, is expected to double by 2060 reaching 98 million and creating demands for better healthcare. While the number of co-occurring diseases increase in the aging population, Alzheimer’s and cancer have been reported to be inversely related – a lower than expected probability of the secondary disease after the primary disease diagnosis. This fueled our interest in exploring genetic variation that is responsible for the inverse relationship between AD and cancer. Rationale & Hypothesis Age is a risk factor for both AD and cancer, and our goal was to compare late-onset AD with two most prevalent age-related cancers – breast and prostate cancer. We hypothesize that harmonizing the age to study the cross-phenotypic effects of genetic variants between AD and cancer against a common control group will identify genetic variants that contribute to their inverse relationship. Methods Genomic SNP data from ADNI (Alzheimer’s Disease Neuroimaging Initiative), ADGC (Alzheimer’s Disease Genetics Consortium) and BPC3 (Breast and Prostate Cancer Care Consortium) which contained 757, 6065, and 11893 individuals respectively included genotypes for up to 700,000 SNP markers. Standard quality control measures were implemented. Individuals with age of disease onset between 60- 80 years were included, and Bayesian multinomial regression was used to compare cases (AD and cancer) against controls in a two-stage replication study. Results A total of 4 SNPs that replicated in the two study stages. In males, two risk loci were significant with opposite odds ratios – rs2075650 mapped to TOMM40 , and an intergenic SNP- rs4298154 on chr 4. Since TOMM40 is near APOE region, we conditioned on APOE SNPs – rs429358 and rs769449, to identify secondary hits. 8 SNPs in the MARK4 region were significant with the inverse hit. In females, rs2075650 was also significant, and conditional analysis resulted in variants in CLPTM1. A non-coding SNP in the VDAC1/ FSTL4 region was also replicated in females. Conclusion Our novel approach has identified four genic regions that have cross-phenotypic effects when comparing AD and cancer: TOMM40/APOE, MARK4, CLPTM1, and VDAC/FSTL4. These genes have been previously implicated independently in AD and cancer and are known to be involved in mitochondrial pathways; however, this is the first study to directly demonstrate that genetic variability in these genes underlies the inverse comorbidity of AD and cancer.Item Assessing Changes to the Lower GI Tract Microbiome in Response to Neglect-related Early Life Stress Exposure(2024-03-21) Choe, Jamie; Donkor, Michael; Zhang, Yan; Gorham, Isabelle; Allen, Michael; Phillips, Nicole; Jones, HarlanGrowing evidence supports exposure to early life stress (ELS) is associated with alterations in the developing immune system and increases the risk for chronic health conditions. It is widely understood that alterations to the gut microbiome can occur from exposure to various environmental factors, including diet and stress. Early life malnutrition is a form of neglect-related ELS that refers to states of both under- and over-nutrition in which a child may have insufficient intake of one or more nutrients due to an imbalanced diet. Malnutrition during childhood is a public health concern with significant health ramifications. Recent research shows the gut microbiome is intimately involved with immune system development—especially during early life when the immune system is being trained. In the present study, we use a modified version of the maternal separation with early weaning (MSEW) model to study the impact of physical neglect and malnutrition on the gut microbiome in mice. Conditions of neglect-related stress were simulated based on scheduled dam-pup separation (physical neglect) and a high carbohydrate early-wean diet (malnutrition). C57BL/6J mice were bred in-house and ELS pups were subjected to: (1) daily dam-pup separation on postnatal days (PD) 2-13 and/or (2) early weaning (EW) to a high carbohydrate diet on PD14-21. All tissues and stool samples were collected on PD21 for analysis. Pups exposed to MSEW or EW alone were assessed separately. 16S rRNA gene sequencing revealed the neglect-related ELS condition, as described under the present model, led to significant shifts in the predominate species in the lower GI tract microbial community. ELS-mediated shifts included increased Bacteroides and Enterococcus and were accompanied by decreased Lachnospiraceae. RTqPCR of bilateral adrenal glands revealed gene expression changes in key enzymes for stress response pathways, namely those implicated in the synthesis of adrenal glucocorticoids. These results demonstrate ELS-mediated dysbiosis can be observed at PD21 under the present model. Our findings at the PD21 timepoint reveal acute changes to the gut microbiome in the context of ELS and characterizes the baseline microbial community in the lower GI tract.Item Assessment of Mitochondrial DNA Damage in Cognitive Impairment via NGS: Health Disparities in Mexican Americans(2022) Reid, Danielle; Barber, Robert C.; Sun, Jie; Thorpe, Roland; Zhou, Zhengyang; Phillips, NicoleMexican Americans (MAs) are the fastest growing subpopulation in the US, and as age increases, this population will be disproportionately affected by age-related diseases such as Alzheimer's disease (AD). Diabetes, stroke, depression, and obesity are common risk factors for developing cognitive impairment (CI) and may be of particular relevance to MAs due to their increased prevalence. MtDNA damage has been implicated in AD, and since metabolic comorbidities are more common in MAs, mtDNA damage and mitochondrial dysfunction may be related to the increased burden and earlier age-of-onset among MAs. Mitochondrial dysfunction can induce oxidative damage to guanosine (8oxoG) and cause DNA deletions, both of which have been well-documented in AD. The mitochondrial genome is particularly vulnerable to DNA damage, and age-associated decline in mitochondrial function results in accumulating reactive oxygen species capable of damaging essential biomolecules. We hypothesize that MAs incur mtDNA damage at an elevated rate due to increased comorbidity burden altering mitochondrial function. MtDNA from buffy coat and plasma samples of participants enrolled in the Texas Alzheimer's Research Care and Consortium were amplified using the RepliG mtDNA Amplification kit and were sequenced via NexteraXT on Illumina NextSeq. Somatic variants indicative of oxidative DNA damage and the commonly observed 5kb deletion were quantified in both the buffy coat mtDNA and ccf-mtDNA. These data were analyzed for association with CI and T2D in both the NHW and MA populations. Further, haplogroup-associated risk for mtDNA damage and ccf-mtDNA status was assessed. Our preliminary findings suggest clinical implications of oxidative mtDNA damage as a risk factor for CI specifically in MA females. These data highlight ethnic/racial differences in oxidative burden which may elucidate sex-specific mechanisms contributing to the manifestation of age-related disease etiology as AD, and the results may ultimately inform precision-based approaches to design therapeutics for mitigating AD disparities in the MA population.Item Association between everyday perceived discrimination and cognitive function as mediated by depression in diverse populations: A HABS-HD Study(2024-03-21) Mendoza, Edna Patricia; Nolan, Emma; Phillips, Nicole; Barber, Robert; O'Bryant, Sid; Zhou, ZhengyangPurpose: Previous research suggests that perceived discrimination is associated with cognitive function impairment, and such association is mediated by depression. With minority populations continuously growing, it is crucial to investigate such relationships in diverse populations. This study aims to examine and compare the above relationships among non-Hispanic white (NHW), Mexican American (MA), and African American (AA) participants. Method: A sample size of 1,129 participants (640 AAs, 248 NHWs, 241 MAs) aged 50+ came from the Health and Aging Brain Study – Health Disparities (HABS-HD). Structural equation modelling (SEM) was conducted to explore the effect between perceived discrimination, measured by the Everyday Discrimination Scale mean score, and cognitive function, measured by the Mini Mental State Examination (MMSE) Score. The mediation effect of depression, measured by the Geriatric Depression Scale total score, was evaluated by the indirect effect estimate using SEM. Result: Everyday perceived discrimination negatively influenced cognitive function, and the effect was mediated by depression across the three populations (β= -0.15, 95% CI = [-0.22, -0.08]). When stratified, the mediation effect of depression on the association between discrimination and cognitive function remained significant for NHW (β= -0.37, 95% CI = [-0.60, -0.15]) and MA (β = -0.27, 95% CI = [-0.50, -0.05]). However, such mediation effect was not observed for the AA population. Conclusion: Depression mediates the link between everyday discrimination and cognitive decline, but differences between racial/ethnic groups underscore the need for further research into underlying mechanisms among minority groups, including Mexican American and African American populations. Depression interventions may mitigate negative cognitive effects from discrimination. Tailoring such interventions by race/ethnicity and targeting at-risk groups could optimally promote cognitive health.Item CELL-FREE MEMBRANE-BOUND AND MEMBRANE-UNBOUND MITOCHONDRIAL DNA IN MATERNAL CIRCULATION IN PREECLAMPSIA(2021) Cushen, Spencer; Ricci, Contessa; Bradshaw, Jessica L.; Silzer, Talisa; Blessing, Alexandra M.; Sun, Jie; Scroggins, Sabrina; Santillan, Mark; Santillan, Donna; Phillips, Nicole; Goulopoulou, StylianiPURPOSE: Cell-free circulating mitochondrial DNA (CFCmtDNA) is a damage-associated molecular pattern (DAMP) that activates Toll-like receptor-9 (TLR-9). Previous studies suggested that CFCmtDNA may be a potential pathogenic trigger or a contributor to the maintenance of preeclampsia. The main objectives of this study were 1) to determine absolute concentrations of CFCmtDNA, in membrane-bound and -unbound states, independent of nuclear DNA (nDNA) changes, in cases with preeclampsia and healthy controls and 2) to implement a penalized regression analysis to establish the contribution of CFCmtDNA to preeclampsia diagnosis and its interaction with commonly collected patient characteristics. METHODS: Plasma CFCmtDNA (MT-ND5 gene) concentrations were quantified using an absolute quantification protocol. DNase I concentrations in maternal plasma were measured using an enzyme-linked immunosorbent assay and TLR-9 activity was monitored using SEAP reporter 293 cells expressing the human TLR-9 gene. RESULTS: Concentrations of CFCmtDNA were reduced in preeclampsia compared to healthy controls both in lysis buffer-treated samples (P=0.02) and in samples not treated with lysis buffer (P< 0.0001). Even though CFCmtDNA concentrations were reduced, plasma from women with preeclampsia induced greater TLR-9 activation than plasma from gestational age matched controls (P< 0.01). Multivariate analysis showed that high concentrations of nDNA and DNase I, a prior history of preeclampsia, and a lower concentration of CFCmtDNA are predictors of preeclampsia diagnosis. CONCLUSIONS: In conclusion, our data demonstrate an increased immunostimulatory potential of CFCmtDNA and upregulation of DNA degradation mechanisms in women with preeclampsia at the third trimester.Item Characterization of Mitochondrial DNA Damage in Complex Disease Using Two Different NGS Platforms(2021) Reid, Danielle; Phillips, Nicole; Barber, Robert C.; Blessing, Alexandra M.Purpose: The Hispanic/Latinx aging (65+) population is expected to increase through 2060 causing the number of Alzheimer's Disease (AD) cases in the Hispanic/Latinx population to quadruple. Several risk factors for developing cognitive impairment are prevalent among Mexican Americans (MAs), although the etiology of these associations remains unclear. Age-associated decline in mitochondrial function results in accumulation of reactive oxygen species (ROS) capable of damaging essential biomolecules, including DNA. The mitochondrial genome is particularly vulnerable to DNA damage, which has a strong correlation with AD pathology. Developing an improved method to assess mitochondrial oxidative damage may help resolve the potential association between abnormal mitochondrial function as indicated by oxidative DNA damage in cognitively impaired MAs. Oxidative damage to guanine (G) forming 8oxoG, is one of the most prevalent DNA lesions. Current methods for detection are limited and lack reproducibility. Lifestyle and/or metabolic health may contribute directly to age-related neurodegeneration. Methods: We aim to investigate the mutational load indicative of oxidative DNA damage in MAs compared to non-Hispanic white (NHW) participants in a human AD cohort, TARCC, who were diagnosed with AD, type-2 diabetes (T2D), and comorbidity (AD/T2D) using Illumina-based NGS. Additionally, we propose nanopore sequencing technology as an improved alternative to current detection/quantification methods. Results: We describe preliminary proof-of-concept results and future applications of this method to analyze mtDNA damage in participants of TARCC. Conclusion: Investigation of oxidative DNA damage may aid our understanding of the differences in manifestation of age-related dementia in MAs.Item Circulating Cell-free Mitochondrial DNA In Normal Human Pregnancy and In Experimental Preeclampsia(2017-03-14) Phillips, Nicole; Sprouse, Marc; Jarvis, Sara; Okada, Yoshiyuki; Morton, Jude; Davidge, Sandra; Fu, Qi; Goulopoulou, Styliani; Chaudhari, SarikaBackground: Mitochondrial DNA (mtDNA) is a damage-associated molecular pattern (DAMP) with potent immunogenic and inflammatory properties. Circulating cell-free mtDNA is increased in various inflammatory conditions associated with intense cell apoptotic processes. Pregnancy is characterized by systemic inflammation and placental apoptosis, which increase with advancing gestational age. The temporal changes of cell-free mtDNA during healthy pregnancy and in pregnancies with preeclampsia are unknown. Hypothesis: Circulating cell free mtDNA increases with gestational age in pregnant women and these changes positively correlate with maternal cardiovascular responses and neonatal biometrics. In a rat model of preeclampsia, circulating mtDNA is increased compared to normotensive control rats. Methods: Normal Human Pregnancy: Maternal blood samples were collected at early pregnancy (≤ 8 weeks of gestation), late pregnancy (32-36 weeks), and postpartum (6-10 weeks after delivery) in healthy, normotensive, pregnant women (n=21). Experimental Model of Preeclampsia: Reduced uterine perfusion pressure (RUPP) was surgically induced in pregnant rats on gestational day (GD) 14. Maternal blood samples were collected from RUPP rats (n=11) and control rats (Sham, n=11) on GD20. Absolute real-time PCR quantification of mtDNA was performed on whole genomic extracts from maternal human and rat sera using TaqMan® probes and chemistry. Results: Normal Human Pregnancy: Circulating mtDNA in late pregnancy were greater compared to early pregnancy (0.02 ± 1.2 pg/μL vs. 0.04 ± 1.2 pg/μL, p=0.04) and remained elevated post-partum (0.03 ± 1.2 pg/μL). Both blood pressure and heart rate increased from early to late pregnancy and decreased postpartum (pExperimental Model of Preeclampsia: RUPP rats had increased circulating mtDNA as compared to the sham group (0.30 ± 0.04 copy number/µL vs. 0.18 ± 0.04 copy number/µL, p=0.03). Conclusions: In normal pregnant women, circulating mtDNA change with advancing gestational age and may reflect rates of placental cell apoptosis. In a rat model of preeclampsia associated with placental ischemia, circulating cell free mtDNA is elevated in late pregnancy. The temporal changes in mtDNA in preeclampsia and their functional role in normal and preeclamptic pregnancies need to be further evaluated.Item Circulating mitochondrial DNA: New indices of type 2 diabetes-related cognitive impairment in Mexican Americans(PLoS, 2019-03-12) Silzer, Talisa K.; Barber, Robert C.; Sun, Jie; Pathak, Gita A.; Johnson, Leigh A.; O'Bryant, Sid E.; Phillips, NicoleMitochondrial function has been implicated and studied in numerous complex age-related diseases. Understanding the potential role of mitochondria in disease pathophysiology is of importance due to the rise in prevalence of complex age-related diseases, such as type 2 diabetes (T2D) and Alzheimer's disease (AD). These two diseases specifically share common pathophysiological characteristics which potentially point to a common root cause or factors for disease exacerbation. Studying the shared phenomena in Mexican Americans is of particular importance due to the disproportionate prevalence of both T2D and AD in this population. Here, we assessed the potential role of mitochondria in T2D and cognitive impairment (CI) in a Mexican American cohort by analyzing blood-based indices of mitochondrial DNA copy number (mtDNACN) and cell-free mitochondrial DNA (CFmtDNA). These mitochondrial metrics were also analyzed for correlation with relevant neuropsychological variables and physiological data collected as indicators of disease and/or disease progression. We found mtDNACN to be significantly decreased in individuals with CI, while CFmtDNA was significantly elevated in T2D; further, CFmtDNA elevation was significantly exacerbated in individuals with both diseases. MtDNACN was found to negatively correlate with age and fatty acid binding protein concentration, while positively correlating with CFmtDNA as well as CERAD total recall score. Candidate gene SNP-set analysis was performed on genes previously implicated in maintenance and control of mitochondrial dynamics to determine if nuclear variants may account for variability in mtDNACN. The results point to a single significant locus, in the LRRK2/MUC19 region, encoding leucine rich repeat kinase 2 and mucin 19. This locus has been previously implicated in Parkinson's disease, among others; rs7302859 was the driver SNP. These combined findings further indicate that mitochondrial dysfunction (as assessed by proxy via mtDNACN) is intimately linked to both T2D and CI phenotypes as well as aging.Item Comparing Methylation of CRP and IL-6 Associated Genes in Cognitively Impaired Mexican Americans to Non-Hispanic Whites(2024-03-21) Sotelo, Joseph; DeLeon, Justin; Housini, Mohammad; Phillips, Nicole; Barber, RobertPurpose: A large pool of literature shows that Alzheimer’s Disease (AD) results from inflammatory processes and neuronal loss via tau proteins and amyloid-β plaques. Mexican Americans are among those with the highest risk of developing Alzheimer’s and research into the epigenetics of this association is lacking. Methylation alterations are influenced by both genetic and lifestyle factors, which can help us identify the root cause of mild cognitive impairment, a precursor for Alzheimer’s Disease. C-reactive protein and interleukin 6 are well known for their roles in measuring systemic inflammation. This study seeks to explore if there is a significant difference between DNA methylation of CpG Islands for CRP and IL6-associated genes in Cognitively Impaired Mexican Americans (MA) and Non-Hispanic Whites (NHW). Methods: Participants were selected from the Texas Alzheimer’s Research and Care Consortium (TARCC) database. The final cohort (n = 551) consisted of 252 NHW (143 normal cognition (NC), and 109 cognitively impaired (CI)) and 299 MA participants (177 NC and 122 CI). Each participant underwent neurocognitive tests such as Clinical Dementia Rating (CDR) and Mini-Mental State Exam (MMSE) to determine cognitive status. Methylation at CpG sites was measured by array probes as beta values with 0=unmethylated, to 1=fully methylated site. CpG sites associated with CRP are cg06126421, cg10636246, cg18181703, cg19821297, and cg25325512. CpG sites associated with IL6 are cg12929678, cg17412005, cg19638572, cg20789595. Any differences between cognitively impaired participants and normal controls were assessed using the standard two-sample t-test assuming unequal variances in Rstudio. Linear Regression was performed in Rstudio, and covariates that were adjusted for include age, sex, education level (in years), APOE ɛ4 allele status, CD8 T-cells, CD4 T-cells, natural killer cells, B-cells, monocytes, and neutrophils. Results: In Mexican Americans, the CRP-associated sites showed: cg25325512 (gene FGD2) with significant hypomethylation in the CI group (p=0.0003). Cg19821297 (gene DNASE2) with significant hypomethylation in the CI group (p=0.015). Cg10636246 (genes AIM2 & IF116) had significant CI group hypomethylation (p=0.02). In Non-Hispanic Whites, one CRP-associated site showed significant hypermethylation in the CI group, cg06690548 (gene TUBB) (p=0.026). In Mexican Americans, the IL6-associated sites showed: cg17412005 (gene MUTYH & TOE1) with significant hypomethylation in the CI group (p=0.022). Cg19638572 (gene RASSF5) with significant hypermethylation in the CI group (p=0.013). Cg20789595 (gene ADCY5) with significant hypomethylation in the CI group (p<0.001). In Non-Hispanic Whites, zero IL6-associated sites showed any significant methylation between the CI and NC groups. Conclusion: Epigenetics related to AD is still being further investigated. This study suggests an association between hypomethylated CRP and IL6 genes and cognitive impairment in the Mexican American population. Limitations in this study include a limited number of CpG sites investigated, as well as possible comorbidities that should be adjusted for. There were also an unequal number of MA to NHW participants. Further studies should adjust for inflammatory comorbidities, such as metabolic syndrome, and further investigation is warranted into the FGD2 gene and ADCY5 gene as they were strongly correlated with hypomethylation in the cognitively impaired Mexican American population.Item Detecting and Quantifying Oxidative DNA Damage using MinION Nanopore Sequencing(2018-05) Blessing, Alexandra M.; Phillips, Nicole; Planz, John; Allen, Michael; He, ShaoqingA common biomarker of damaged DNA, particularly mitochondrial DNA, 8-oxoguanine (8-oxoG) has been identified as a possible contributor to neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, preeclampsia, as well as type 1 and type 2 diabetes. Numerous methods have been developed to detect oxidative damage within the genome, including but not limited to immunological techniques, quantitative-polymerase chain reaction (qPCR), and in situ imaging. This study explores nanopore sequencing using the MinION Nanopore (Oxford Nanopore Technologies, Oxford, UK) as a more sensitive method of 8-oxoguanine detection, providing proof-of-concept for model training as well as preliminary model development.Item Epigenetic Changes of Nuclear-Encoded Oxidative Phosphorylation Genes and Cognitive Function: A Study of Mexican Americans and Non-Hispanic Whites(2024-03-21) Swami, Anjana; Daniel, Ann Abraham; Silzer, Talisa; Sun, Jie; Barber, Robert; Phillips, NicolePurpose: There is a higher prevalence of metabolic disease and Alzheimer’s Disease (AD) in Mexican Americans (MA). Despite this data, there has been minimal research done on the methylation status of genes involved in mitochondrial oxidative phosphorylation (OXPHOS)/cellular metabolism and how this influences the risk for developing cognitive impairment (CI). Methods: Results were derived from 299 MAs and 252 non-Hispanic Whites (NHW), all of whom were participants of the Texas Alzheimer’s Research and Care Consortium (TARCC). Themethylation status of CpG sites was assessed by running peripheral blood samples on the InfiniumMethylationEPIC BeadChip array. Results: Based on a Bonferroni adjusted alpha of7.36485 x 10⁶, six differentially methylated sites were significant in MAs: cg07470503, cg10057295, cg13823120, cg26891598, cg21490662, and cg17904988. All the sites were hypomethylated in CI/AD cohorts compared to NC except for cg26891598. There were no sites of significance in NHWs. Conclusions: The strongest association with CI/AD within the MA cohort was at cg07470503, with a p-value of 1.00 x 10⁶ in MAs. This CpG site is found within the DGUOK gene. The DGUOK gene is responsible for making the enzyme deoxyguanosine kinase, which is needed to properly create mitochondrial DNA; a dysfunctional gene leads to impaired mitochondrial function that could decrease the efficiency of OXPHOS. The abnormal cellular metabolism that ensues could set up the foundation for neurodegeneration to occur. Moving forward, the cg07470503 site could serve as a marker to identify the risk of metabolic disease and consequent CI/AD in MA patients.Item Epigenetic Risk Factors for Mild Cognitive Impairment, Alzheimer’s Disease and Metabolic Dysfunction in Mexican Americans(2018-03-14) Silzer, Talisa; Sun, Jie; Phillips, Nicole; Johnson, Leigh; O'Bryant, Sid; Barber, Robert C.; Abraham Daniel, AnnPurpose: Alzheimer’s is the most common form of dementia and the 5th leading cause of death for those over 651. The population of Mexican American elders will grow seven-fold by 20502 with rates of mild cognitive decline (MCI) and Alzheimer’s disease (AD) increasing exponentially1. Mexican Americans are diagnosed with MCI and AD at younger ages than non-Hispanic whites3; 4. In addition, Mexican Americans who are diagnosed with AD are 1) less likely to carry the ApoEε4 genotype3-5., 2) suffer a greater burden of type 2 diabetes3; 6, 3) experience greater metabolic-related cognitive decline7; 8 and 4) display a proteomic signature of AD that is heavily metabolic in nature4; 9, compared to non-Hispanic whites, whose proteomic signature for AD is dominated by inflammatory proteins. We hypothesized that differentially methylated regions of DNA (DMRs) are associated with age at onset of cognitive decline (MCI/AD) and metabolic dysfunction (metabolic syndrome/type 2 diabetes) in Mexican Americans. Methods: To test this hypothesis, we assayed genomic DNA methylation in samples from 14 female Mexican American participants enrolled in the Health and Aging Brain study in Latino Elders (HABLE). Participants were diagnosed with cognitive decline (n=4), metabolic dysfunction (n=3), both (n=4), or as a control (n=3). We isolated DNA from leukocytes and bisulfite treated the samples before running them on an Illumina MethylationEPIC chip in accordance with manufacturer’s recommendations to assay genomic DNA methylation. Results: Several interesting biological pathways showed significantly different methylation status between groups. When the participants were split on cognitive decline, DNA in the amyloid secretase, EGF receptor signaling, PDGF signaling, gonadotropin-releasing hormone receptor and Wnt signaling pathways were significantly hypermethylated in cases. In comparison, analyses based on metabolic dysfunction showed significant DNA hypomethylation in the beta1 and beta2 adrenergic receptor signaling pathways and hypomethylation of the gonadotropin releasing hormone receptor pathway in cases. Conclusions: The etiology of cognitive decline appears to differ between Mexican Americans and non-Hispanic whites. Future work will resolve how dementia risk differs between these and other ethnic groups. The knowledge gained from these studies will be critical to a better understanding of AD pathophysiology and the development of ethnicity-focused AD treatment options. Acknowledgements: Research reported here was supported by the National Institute On Aging of the National Institutes of Health under Award Number R01AG054073. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The research team also thanks the local Fort Worth community and participants of the Health & Aging Brain Study.Item Epistatic impact of APOE and ACE2 genetic variants on SARS-CoV-2 and RAS dependent blood-brain barrier dysregulation.(2024-03-21) Tate, Amanda; Barber, Robert; Park, Inwoo; Jones, Harlan; Phillips, NicoleCoronavirus disease 2019 (COVID-19) is associated with respiratory and neurodegenerative symptoms, creating a need to understand the additional impact the pandemic might have on neurodegeneration and risk for neurodegenerative diseases, such as Alzheimer’s disease (AD) in aging populations post 2020. The blood-brain barrier (BBB) is an important interface that connects the periphery to the brain through the vasculature. When this protective barrier becomes dysregulated, the brain is vulnerable to infection, neuroinflammation, and cellular stress, which over time can lead to neurodegeneration and cognitive decline. The renin-angiotensin system (RAS) is an important regulator of vasculature via the activity of the hormone angiotensin II (Ang II). As a mediator of vasoconstrictive, oxidation, and inflammatory responses, its prolonged activity can be damaging and promote neurodegeneration at the BBB. Angiotensin-converting enzyme 2 (ACE2) is highly expressed in endothelial cells which cleaves Ang II into less harmful fragments to offset these potentially toxic effects. Genetic variants of ACE2 are increasingly considered risk factors for the development of vascular disorders (e.g. hypertension) due to their role in RAS-mediated dysregulation of body vasculature. Recent genetic studies have identified a relationship between genetic variants for ACE2, an obligate receptor for the severe acute respiratory syndrome coronavirus 2 (SCoV2), and increased risk and or/severity of COVID-19. Alzheimer’s disease (AD) is the greatest neurological risk among aging individuals and can be caused by both environmental & genetic risk factors. The strongest genetic risk factor for AD is variants in the apolipoprotein E gene (APOE), with the ɛ4 allele being associated with increased levels of amyloid β (Aβ), Tau proteins (p-Tau), neuroinflammation, and BBB permeability. A recent study by Wang et al also suggests a correlation between APOE ɛ4 and increased severity of COVID-19, suggesting some interplay between APOE and host factors related to SCoV2 infection. Furthermore, both ACE2 and APOE genetic variants disproportionately impact minority populations, highlighting a need to understand the health disparity of AD and COVID-19 risk across demographic groups. We hypothesize gene interactions (epistatic interactions) between genetic variants in ACE2 and APOE may exacerbate RAS-mediated BBB dysregulation, leading to increased AD phenotypes and SCoV2 neurological dysregulation. To address this hypothesis, we will create endothelial cells, astrocytes, and neurons containing APOE and ACE2 genetic variants of interest. These epistatic cells will be assessed for expression and functional changes related to BBB integrity. Using Ang II treatments and SCoV2 pseudo-virus models, we will assess the impact of COVID-19 and RAS on BBB integrity and functions. This study will help us understand the mechanisms and interplay of genetic risks for AD and COVID-19 related to RAS-mediated BBB dysregulation, potentially highlighting comorbidities among the aging population. By focusing on ACE2 variants disproportionately found in minority populations, we will provide knowledge surrounding two co-morbidities for neurodegeneration and elevate those at the highest risk for developing AD and/or COVID-19. Funding: This work is supported by the Neurobiology of Aging and Alzheimer’s Disease T32 Training Fellowship, and the IMSD Fellowship, Grant # 5 R25 GM125587-05 from the National Institutes of General Medical Sciences (NIGMS).Item EXOSOME PROFILING OF BRONCHIAL LAVAGE FLUID IN A MOUSE MODEL OF SURGERY RESECTION OF BREAST CANCER WITH LUNG METASTASIS(2024-03-21) Marikh, Morad; Brown, Ainsley; Hall, Courtney; Donkor, Michael; Garlotte, Isabelle; Subasinghe, Kumudu; Elkassih, Omar; Jones, Harlan; Phillips, NicoleThe lung serves as a primary site for breast cancer metastasis, carrying profound implications for patient prognoses. About 60% of people diagnosed with metastatic breast cancer have lesions in either the lungs or the bones, with triple-negative breast cancer (TNBC) more likely than other types of breast cancers to metastasize to the lungs. Although current targeted chemo-radiotherapy and surgery result in higher survivorship, studies have documented that such curative treatments may also increase risk of lung metastasis. To date, the causal factors that mediate metastasis in the context of cancer treatments remain elusive. Our long-term goal is that a deeper understanding of the mechanisms that mediate relocation of breast tumor cells from its primary origin to its distal site (e.g., lung) will reveal novel complementary diagnostic and preventative treatments to improve TNBC survivorship. Exosomes, serving as tiny extracellular vesicles within tumor cells and other cells (e.g., immune cells) release diverse biomolecules have been implicated in tumor pathogenesis. Specifically, miRNAs as cargo within exosomes are known to regulate cellular function. miRNAs are small RNA molecules that can bind to messenger RNA (mRNA) and inhibit protein synthesis or promote mRNA degradation. This regulatory function allows miRNAs to modulate the expression of multiple genes involved in various cellular processes and their dysregulation has been implicated in various diseases, including cancer. The objective of this study was to determine the expression of miRNA-200b-3p and miRNA-141-5p as known regulators of lung cancer are influenced by surgical removal of a primary breast cancer. We hypothesized that miRNA-200b-3p and miRNA-141-5p mRNA expression is increased in response to surgery. Using an established model of breast cancer metastasis, exosomes were isolated from the bronchiole alveolar lavage fluid (BALF) of tumor bearing mice and mice in which primary tumors were resected compared to tumor-free mice. Results demonstrated that miRNA-200b-3p was present in both tumor-bearing and non-tumor-bearing mice. In contrast, miRNA-141-5p was not expressed in tumor-bearing, non-tumor-bearing mice, and naïve mice determined by quantitative reverse transcriptase polymerase chain reaction (qrtPCR). In conclusion, as we navigate the intricacies of miRNA dynamics in the lung microenvironment, future studies will involve broadening the miRNA panel and refining exosome recovery techniques. This strategic evolution aims to enhance sensitivity, facilitating the detection of elusive, tumor-derived exosome miRNAs. All studies have been approved by UNTHSC IACUC, approval number #2018-0031. Acknowledgement: This research is partially supported by a grant from the Cancer Prevention and Research Institute of Texas (Award#: RP210046) to Dr. Jamboor K. Vishwanatha and National Institute of Cancer Research of the Health under Award 1 P20 CA233355-01 (Vishwanatha, Jones-Project 1).Item Hypermethylation at CREBBP Is Associated with Cognitive Impairment in a Mexican American Cohort(IOS Press, 2023-03-07) Abraham Daniel, Ann; Silzer, Talisa; Sun, Jie; Zhou, Zhengyang; Hall, Courtney; Phillips, Nicole; Barber, Robert C.BACKGROUND: The aging Mexican American (MA) population is the fastest growing ethnic minority group in the US. MAs have a unique metabolic-related risk for Alzheimer's disease (AD) and mild cognitive impairment (MCI), compared to non-Hispanic whites (NHW). This risk for cognitive impairment (CI) is multifactorial involving genetics, environmental, and lifestyle factors. Changes in environment and lifestyle can alter patterns and even possibly reverse derangement of DNA methylation (a form of epigenetic regulation). OBJECTIVE: We sought to identify ethnicity-specific DNA methylation profiles that may be associated with CI in MAs and NHWs. METHODS: DNA obtained from peripheral blood of 551 participants from the Texas Alzheimer's Research and Care Consortium was typed on the Illumina Infinium® MethylationEPIC chip array, which assesses over 850K CpG genomic sites. Within each ethnic group (N = 299 MAs, N = 252 NHWs), participants were stratified by cognitive status (control versus CI). Beta values, representing relative degree of methylation, were normalized using the Beta MIxture Quantile dilation method and assessed for differential methylation using the Chip Analysis Methylation Pipeline (ChAMP), limma and cate packages in R. RESULTS: Two differentially methylated sites were significant: cg13135255 (MAs) and cg27002303 (NHWs) based on an FDR p < 0.05. Three suggestive sites obtained were cg01887506 (MAs) and cg10607142 and cg13529380 (NHWs). Most methylation sites were hypermethylated in CI compared to controls, except cg13529380 which was hypomethylated. CONCLUSION: The strongest association with CI was at cg13135255 (FDR-adjusted p = 0.029 in MAs), within the CREBBP gene. Moving forward, identifying additional ethnicity-specific methylation sites may be useful to discern CI risk in MAs.Item Imputation Accuracy of Apolipoprotein E ε Alleles in Genome-wide arrays and real-time SNP Genotyping assays(2022) Subasinghe, Kumudu; Garlotte, Isabelle; O'Bryant, Sid E.; Barber, Robert C.; Phillips, NicolePurpose: The vast majority of the established genetic-based risk for late-onset Alzheimer's disease (AD) is attributable to variation within the apolipoprotein E (APOE) gene. This gene, which encodes a protein implicated in various aspects of AD pathology, is characterized by two single nucleotide polymorphisms (SNPs; rs429358 and rs7412) that result in three distinct isoforms (ε4, ε3 and ε2). Most population-based genome-wide association studies to date have identified the APOE ε4 and ε2 alleles as the strongest genetic-based risk and protective factors for AD, respectively. APOE genotype is not only critical for determining disease risk and diagnosis, but also for developing individualized therapeutic strategies. Genotyping via real-time quantitative PCR (qPCR) is the gold standard for APOE isoform determination; however, if genome wide SNP data is available, imputation of APOE (i.e., probabilistic genotyping through inference) may eliminate the need for qPCR genotyping. In this project, we evaluate the concordance of APOE genotypes obtained via qPCR and a genome-wide SNP chip in non-Hispanic White and Mexican American individuals from the Health & Aging Brain among Latino Elders (HABLE) cohort. Method: DNA was extracted from buffy coat samples (n = 1650) on the Hamilton robotic system with the Mag-Bind Blood & Tissue DNA HDQ 96 Kit. qPCR was then performed using the TaqMan Genotyping Kit as per manufacturer's protocol. Results produced via qPCR were then compared to those imputed for rs429358 and directly typed for rs7412 on the Illumina Infinium Global Screening Arrays (GSA) and analyzed with Genome Studio 2.0. Samples with call rates less than 98% were repeated or excluded. Results: Concordance between the APOE genotypes obtained from qPCR and Infinium GSA was 99.32%. Discordance was likely due to poor sample quality and low-frequency imputation errors of rs429358, which may be corrected with more conservative thresholding of the imputed genotype confidence statistics. Conclusion: Genotype imputation from SNPs commonly typed in the APOE region is an effective method for APOE isoform determination, even in Mexican Americans who are more genetically heterogenous due to ancestral admixture; this method may be effectively implemented in large population-based studies of aging and AD.Item Inhibition of Mitochondrial Respiratory Chain Complex I Induces Vascular Endothelial Cell Apoptosis and Release of Mitochondrial DNA(2019-03-05) Cushen, Spencer; Phillips, Nicole; Goulopoulou, Styliani; Riaz, MaryamPurpose: Vascular endothelial oxidative stress is a common feature of preeclampsia, a pregnancy specific hypertensive syndrome with high incidence of maternal and fetal mortality and morbidity. Cellular oxidative stress can lead to cell death, which promotes the release of cellular constituents (e.g. mitochondrial fragments) into the extracellular space. Circulating cell-free mitochondrial DNA (mtDNA) concentrations are increased in pregnant women with preeclampsia. The main objective of this study was to determine the mechanisms by which vascular endothelial cells may contribute to this increase in cell-free mtDNA. The hypothesis was that mitochondrial complex I inhibition results in extrusion of mtDNA from vascular endothelial cells via cell death-dependent mechanisms. Methods: Human umbilical vein endothelial cells (HUVEC, Lonza) were grown to 80-90% confluency before treatment with a mitochondrial complex I inhibitor (Rotenone: 5, 10, 25 μM - 4 h). Immunocytochemistry was used to confirm that HUVEC maintained endothelial cell characteristics. Cell death (apoptotic and non-apoptotic) was quantified using flow cytometry (staining for Annexin V and propidium iodide). mtDNA was measured on total nucleic acid extracts from cell culture supernatants using absolute real-time PCR techniques. Results: Treatment of HUVECs with rotenone increased early apoptosis and late apoptosis/necrosis [5μM (n=7), Veh: 11.16 ± 1.96% vs Rotenone: 14.74 ± 1.96% p=0.0159; 10μM (n=7), Veh: 10.54 ± 1.93% vs Rotenone: 14.83 ± 2.60% p=0.0033; 25μM (n=7), Veh: 10.34 ± 1.85% vs Rotenone: 15.87 ± 3.023% p=0.0002; 1-way ANOVA followed by Sidak’s post-hoc test]. Concentrations of mtDNA in HUVEC supernatant were increased in HUVECs treated with 5 μM of Rotenone [Veh (n=5): 2.45 ± 0.05 pg/mL vs. Rotenone (n=6): 3.65 ± 0.39 pg/mL, p=0.0700; Sidak’s post-hoc test]. Higher concentrations of Rotenone had no effect on concentrations of extracellular mtDNA (p [greater than] 0.84). Conclusions: Mitochondrial oxidative stress due to inhibition of mitochondrial respiratory chain complex I induces vascular endothelial cell death. Extrusion of mtDNA from apoptotic and necrotic endothelial cells may contribute to increased circulating mtDNA concentrations in preeclamptic pregnancies. Future studies will test this hypothesis using integrative pharmacological and physiological approaches.Item Investigating Oxidative Damage: Implications in Cognitive and Metabolic Phenotypes of Alzheimer's Disease in the Mexican American Population(2020) Phillips, Nicole; Barber, Robert C.; Silzer, Talisa; Blessing, Alexandra; Reid, DanielleThe elderly population is rapidly expanding leading to increases in age-related diseases such as cardiovascular disease, metabolic disorders, cancer, and neurodegenerative diseases. The Texas Alzheimer's Research and Care Consortium (TARCC) is a collaborative research effort to identify factors involved in the development and progression of Alzheimer's Disease (AD) in Mexican Americans (MAs). In the MA population, diabetes, depression, stroke, and obesity are common risk factors for developing cognitive impairment. The reasons for the association between cognitive decline and comorbidities remain unclear. Studies have shown correlations between common pathological changes observed in AD and those that are a result of DNA damage. The mitochondrial genome is particularly vulnerable to DNA damage due to its close proximity to reactive oxygen species (ROS). Age associated declines in mitochondrial function results in accumulation of ROS, which are capable of damaging DNA and other essential biomolecules. Oxidative damage to DNA takes many forms, however oxidation of guanine (G) forming 8oxoG, is one of the most prevalent DNA lesions. Currently, the methods for detection of 8oxoG are limited and lack reproducibility due to several reasons. We propose nanopore sequencing technology as an improved alternative to current detection and quantification methods. Here we describe preliminary proof of concept results and discuss future applications of this method for analysis of mtDNA damage in participants of the TARCC cohort. Investigation of oxidative DNA damage may aid our understanding of the differences in manifestation of age-related cognitive decline in Mexican Americans as compared to non-Hispanic whites.
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