Browsing by Author "Barber, Robert C."
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Item A Synthetic Formula Amino Acid Diet Leads to Microbiome Dysbiosis, Reduced Colon Length, Inflammation, and Altered Locomotor Activity in C57BL/6J Mice(MDPI, 2023-11-25) Mancilla, Viviana J.; Braden-Kuhle, Paige N.; Brice, Kelly N.; Mann, Allison E.; Williams, Megan T.; Zhang, Yan; Chumley, Michael J.; Barber, Robert C.; White, Sabrina N.; Boehm, Gary W.; Allen, Michael S.The effects of synthetic, free-amino acid diets, similar to those prescribed as supplements for (phenylketonuria) PKU patients, on gut microbiota and overall health are not well understood. In the current, multidisciplinary study, we examined the effects of a synthetically-derived, low-fiber, amino acid diet on behavior, cognition, gut microbiome composition, and inflammatory markers. A cohort of 20 male C57BL/6J mice were randomly assigned to either a standard or synthetic diet (n = 10) at post-natal day 21 and maintained for 13 weeks. Sequencing of the 16S rRNA gene from fecal samples revealed decreased bacterial diversity, increased abundance of bacteria associated with disease, such as Prevotella, and a downward shift in gut microbiota associated with fermentation pathways in the synthetic diet group. Furthermore, there were decreased levels of short chain fatty acids and shortening of the colon in mice consuming the synthetic diet. Finally, we measured TNF-alpha, IL-6, and IL-10 in serum, the hippocampus, and colon, and found that the synthetic diet significantly increased IL-6 production in the hippocampus. These results demonstrate the importance of a multidisciplinary approach to future diet and microbiome studies, as diet not only impacts the gut microbiome composition but potentially systemic health as well.Item Admixture Effects on Coevolved Metabolic Systems(2016-05-01) Zascavage, Roxanne R.; Planz, John V.; Barber, Robert C.; Clark, Abbot F.Oxidative phosphorylation (OXPHOS) is the primary energy generating system in eukaryotic organisms. Consequently, any malfunctions or disruptions in the pathway significantly impact fitness and health. Errors in energy production have been linked to cancer, Alzheimer’s disease, Parkinson’s disease, various neuropathies, and general aging and health degeneration over time. However, there is a fundamental gap in the understanding of the genetic causes of deficiencies in energy production. The complexes within the OXPHOS pathway are of mixed origin; while most subunit-coding genes are located within the nuclear genome, thirteen are coded for in the mitochondrial genome. There is strong evidence to support coadaptation between the two genomes in these OXPHOS gene regions in order to create tight protein interactions necessary for a functional energetic system. While the effect of separating coevolved alleles is not fully understood, hybrid studies have indicated decreased energy production when combining different ancestral nuclear and mitochondrial backgrounds in various species. This suggests the common human practice of interpopulation matings between ancestrally distinct groupings influences health and relative fitness. The primary hypothesis is that admixture creates maladaptive combinations of nuclear and mitochondrial alleles in the OXPHOS-coding genes that have adverse effects on the efficiency of energy production, leading to a decrease in relative fitness. This dissertation project has: 1) identified the effects of admixture on OXPHOS activity in Mus musculus populations, showing that high admixture leads to significantly lower basal respiration rates; and 2) assessed the genetic composition of the strains of Mus musculus evaluated to identify to cause of the loss of respiration in highly admixed mice. It was determined that there were no genetic anomalies present that could explain the observations, meaning the cause is likely not due to a mutation, but instead an undetected difference, such as cyto-nuclear incompatibility. It is recommended that further energetic and genetic studies be performed to identify the source of the deficiency. Mice obtained from Jackson Laboratories and a previously published genotype dataset [82] were used for experiments. Laboratory experiments included: Liver and heart extraction, tissue preparation and bioenergetics analysis, statistical analysis, and genetic analysis.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 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 Ancestry Informativeness of Alu Markers in Four Populations Relevant for the United States(2015-05-01) D'Auben, Aislinn G.; Chakraborty, Ranajit; LaRue, Bobby L.; Barber, Robert C.Determination of ancestry using DNA markers is an important issue in DNA forensics. The ability to identify an individual’s ancestry could narrow down the pool of possible individuals involved in a crime. Several types of ancestry informative markers (AIMs) have been suggested in the literature. For this study, Alu markers were used for investigating their utility for Caucasian versus African and Caucasian versus Asian ancestry determinations. Three measures of AIMs were calculated for 42 Alu markers. Rank correlations of these three measures were used for investigating if a smaller number of top-ranked loci can improve ancestry determination. The Alu markers chosen for this study were less informative than anticipated but did show potential for ancestry estimation when all 42 markers were used together.Item Androgen Modulation of CNS During Chronic Intermittent Hypoxia(2018-05) Snyder, Brina D.; Cunningham, Rebecca L.; Barber, Robert C.; Cunningham, J. Thomas; Schreihofer, Derek A.; Planz, John V.The underlying causes of age-related neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease, are unknown. It is likely conditions which contribute to an abundance of oxidative stress throughout life renders an individual more susceptible to late-life neurodegenerative processes. Sex differences are observed in the onset and prevalence of these diseases, suggesting estrogens and androgens influence these processes. This study investigates the early role of androgens under a known oxidative stressor, sleep apnea, which frequently goes untreated in the clinical population but is but is associated with an increased risk of late-life neurodegeneration. The hypoxic events of sleep apnea can be modeled in rats by the use of chronic intermittent hypoxia (CIH). Male rats are more susceptible to hypertensive effects of CIH, a key characteristic of sleep apnea. After one week of CIH treatment, they also exhibit oxidative stress and inflammation in circulation and in brain nuclei associated with early stages of Parkinson's disease or Alzheimer's disease. This led to the hypothesis that oxidative stress and inflammation would be associated with behavior deficits and these effects are mediated by androgens. Results show that oxidative stress and inflammatory dysregulation can be prevented by testosterone, but are highly exacerbated by testosterone's non-aromatizable metabolite, dihydrotestosterone (DHT). Administration of DHT also resulted in significant memory impairments under CIH. In the central nervous system, DHT significantly altered oxidative stress and pro-inflammatory signals, which may underlie its detrimental actions in an oxidative stress environment. There was also evidence of hypothalamic-pituitary-adrenal axis dysregulation, which can influence testosterone and circadian rhythms. These findings have broad implications for clinical populations with conditions which chronically increase oxidative stress and inflammation, while at the same time alter endocrine function. Conditions, such as untreated sleep apnea, may pose a latent risk for neurodegeneration and should be addressed early to prevent later detrimental effects.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 Astrocyte-mediated HIV-1 Tat neurotoxicity and its molecular mechanisms: astrocyte activation and impaired neurogenesis(2016-08-01) Fan, Yan; He, Johnny J.; Ghorpade, Anuja; Barber, Robert C.Human immunodeficiency virus type 1 (HIV-1) invasion of the central nervous system (CNS) often causes motor and cognitive dysfunction, which is termed HIV-associated neurocognitive disorders (HAND). Although the introduction of combination antiretroviral therapy (cART) has effectively suppressed viral replication, improved immune function and increased life expectancy among HIV-infected individuals, it has failed to provide complete protection from HAND or to reverse the disease. HIV-1 Tat protein is a major pathogenic factor in HAND. Studies including ours have demonstrated that Tat is taken up by HIV-1 uninfected brain cells and alters the function of these cells, especially astrocytes, neurons and neural progenitor cells. However, the underlying mechanisms are still unclear. There are two parts to my dissertation research. In the first part, we determined the roles of signal transducer and activator of transcription 3 (STAT3) in Tat-induced glial fibrillary acidic protein (GFAP) transactivation. Astrocytes, the most abundant cells in the brain, not only provide the scaffold support in the brain, but also are essential for maintaining the homeostasis of the brain. GFAP is the specific molecular marker for astrocytes, but it also modulates astrocyte function both physiologically and pathologically. In this study, we first showed that STAT3 expression and phosphorylation led to significant increases in GFAP transcription and protein expression. Then we determined that Tat expression was associated with increased STAT3 expression and phosphorylation in Tat-expressing astrocytes and HIV-infected astrocytes. In addition, we showed that GFAP, Egr-1 and p300 transcription all showed positive response to STAT3 and its phosphorylation. Moreover, knockdown of STAT3 resulted in significant decreases in Tat-induced GFAP and Egr-1 transcription and protein expression. Taken together, these findings show that STAT3 is involved in and acts upstream of Egr-1 and p300 in the Tat-induced GFAP transactivation cascade and suggest important roles of STAT3 in controlling astrocyte proliferation and activation in the HIV-infected CNS. In the second part of the dissertation research, we took advantage of the doxycyclineinducible and astrocyte-specific HIV-1 Tat transgenic mice (iTat) and determined the relationship between Tat expression and neurogenesis. Tat expression in astrocytes was associated with detection of fewer neuron progenitor cells (NPC), fewer immature neurons and fewer mature neurons in the dentate gyrus of the hippocampus of the mouse brain. In vitro NPC-derived neurosphere assays showed that Tat-containing conditioned media from astrocytes and recombinant Tat protein inhibited NPC proliferation and migration and altered NPC differentiation, while immunodepletion of Tat from Tat-containing conditioned media or heat inactivation of recombinant Tat abrogated those effects. Notch signaling downstream gene Hes1 promoter-driven luciferase reporter gene assay and Western blotting showed that recombinant Tat and Tat-containing conditioned media activated Hes1 transcription and protein expression, which were abrogated by Tat heat inactivation, immunodepletion, and cysteine mutation at position 30. Moreover, Notch signaling inhibitor DAPT significantly rescued Tat-impaired NPC differentiation in vitro and neurogenesis in vivo. Taken together, these results show that Tat adversely affects NPC and neurogenesis through Notch signaling and point to the potential of developing Notch signaling inhibitors as HAND therapeutics.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 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 Common Lung Microbiome Identified among Mechanically Ventilated Surgical Patients(PLOS, 2016-11-29) Smith, Ashley D.; Zhang, Yan; Barber, Robert C.; Minshall, Christian T.; Huebinger, Ryan M.; Allen, Michael S.The examination of the pulmonary microbiome in patients with non-chronic disease states has not been extensively examined. Traditional culture based screening methods are often unable to identify bacteria from bronchoalveolar lavage samples. The advancement of next-generation sequencing technologies allows for a culture-independent molecular based analysis to determine the microbial composition in the lung of this patient population. For this study, the Ion Torrent PGM system was used to assess the microbial complexity of culture negative bronchoalveolar lavage samples. A group of samples were identified that all displayed high diversity and similar relative abundance of bacteria. This group consisted of Hydrogenophaga, unclassified Bacteroidetes, Pedobacter, Thauera, and Acinetobacter. These bacteria may be representative of a common non-pathogenic pulmonary microbiome associated within this population of patients.Item Epigenetic alterations in Brain tissue and Alzheimer's Disease(2015-03) Shewale, Shantanu J.; Barber, Robert C.; Planz, JohnBackground: Epigenetic factors such as methylation of DNA have shown to impact the phenotype of a cell and irregular methylation of DNA has been correlated with numerous diseases. DNA methylation has been shown to impact the expression of certain genes associated with AD. Methods: A novel method, methylated DNA immunoprecipitation (MeDIP), is used to study genome wide methylation patterns via high throughput sequencing to assess DNA methylation patterns in brain tissue from individuals diagnosed with AD (N=12) and age matched controls (N=12). MeDIP isolation facilitates an unbiased methylation analysis of the entire human genome by enriching samples for methylated DNA fragments. The MethylMiner kit (Life Technologies) was utilized to precipitate methylated DNA, which was sequenced on the Illumina Hi-seq 2500. In addition, another aliquot will undergo MeDIP and bisulfite treatment. This will allow analysis of methylated cytosines at single base pair resolution across the entire genome. In addition, RNA and miRNA-seq was performed on the Illumina Hi-seq 2500. RNA-seq information obtained provides additional insight on epigenetic impacts on miRNA and RNA levels. Results: Sequence data reveal a genome wide methylation pattern profile, along with gene expression changes that differentiate case from control participants. Conclusions: These data provide insight and help explain a portion of the missing heritability that has yet to be characterized for AD.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 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 Diversity in Sioux Indians of South Dakota with 21 Autosomal STR Loci and Their Forensic Utility(2015-05-01) Grubb, Nicole C.; Chakraborty, Ranajit; Budowle, Bruce; Barber, Robert C.Short Tandem Repeats are the current standard markers in DNA Forensics. They have been extensively studied in cosmopolitan populations, but not so in small isolated populations, which tend to have unique societal structures impacting their genetic diversity. This project examined 21 autosomal STR loci in Sioux Indians and used NIST data of the same loci on four major U.S. populations to make the comparisons. The study concluded that the Sioux Indians have a reduced genetic diversity at these loci, but this reduction did not compromise their utility for forensic applications for human identification. Genetic diversity analyses of individual Sioux Indian tribes suggested that a pooled database is appropriate for DNA forensic applications.Item Highly Informative Short Tandem Repeat Markers for Enhanced DNA Mixture Deconvolution(2018-08-01) Novroski, Nicole M. M.; Budowle, Bruce; Barber, Robert C.; LaRue, Bobby L.DNA typing in forensic genetics relies on amplification of short tandem repeat (STR) markers using the polymerase chain reaction (PCR), subsequently allele sizes are determined for each locus, using capillary electrophoresis (CE) and fluorescent detection. The resulting profiles are compared to reference sample profiles or to query existing profiles, such as those stored in the FBI Combined DNA Index System, to develop investigative leads to help solve crimes. The success of commercial STR kits to facilitate analysis of challenging samples has led to a demand to analyze increasingly complex DNA mixtures. Low quantity/low quality DNA samples have become commonplace in casework, but the interpretation of the resultant DNA profiles continues to remain challenging. Massively parallel sequencing (MPS) for typing forensically-relevant STR loci has dramatically enhanced the ability to identify allele diversity due to sequence variation within STR repeat and flanking regions. Sequence variation within the currently utilized STR loci for forensic genetic analysis is quite large. However, recent studies have demonstrated that some of the current core CODIS loci are devoid of repeat and/or flanking region sequence variation, minimizing the relative information via MPS for these STRs. Thus, novel STRs with increased sequence variation should be sought to facilitate mixture deconvolution. The primary goal of this research was to identify and characterize STR genetic variation, which in turn would allow for the development of a novel panel of highly polymorphic STR markers (referred to as the STR DECoDE panel; STR DNA EnhanCed DEconvolution panel) that is capable of deconvolving simple to complex DNA mixture samples better than current systems. A list of candidate STRs was generated by mining the 1000 Genomes Project using the criteria of 1) a repeat size of at least 4 nucleotides; 2) a minimum of 80% locus heterozygosity; and 3) generally an allele length spread of 10 nominal alleles or less. A preliminary panel of 248 candidate markers was designed, and a bioinformatics pipeline for MPS was created and implemented to assess the analytical performance and biological properties of each STR. The STR DECoDE panel is comprised of 73 of the 248 STRs that displayed the highest heterozygosity. This panel was compared to the current core CODIS loci regarding an ability to resolve in silico two-person mixtures from 443 population samples comprising three US populations. Additionally, each of the 73 loci was extensively characterized for its underlying genetic variation, and population genetic analyses were performed. The results of this dissertation research indicate that the STR DECoDE panel improves upon current mixture deconvolution efforts by employing markers that allow for better allele resolution of component contributors in a mixed DNA sample. The DECoDE panel loci offer a substantial degree of diversity compared with the current core CODIS STR loci used for forensic identity typing. In turn, use of this panel could facilitate complex downstream statistical modeling (probabilistic genotyping) and subjective interpretation that are currently utilized for analysis of DNA mixture samples in forensic laboratories. Finally, integration of DECoDE STR loci into current multiplexes will allow the field of forensic genetic investigation to increase the number of resolved genotypes in mixed samples being compared to reference and suspect profiles, and expand the DNA database by increasing the number of samples uploaded. The benefit to society from this revolutionary application will be an increase in the number of investigative leads and the overall resolution of more crimes.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 Identification of long non-coding RNA-related and -coexpressed mRNA biomarkers for hepatocellular carcinoma(BioMed Central Ltd., 2019-01-31) Zhang, Fan; Ding, Linda; Cui, Li; Barber, Robert C.; Deng, BinBackground: While changes in mRNA expression during tumorigenesis have been used widely as molecular biomarkers for the diagnosis of a number of cancers, the approach has limitations. For example, traditional methods do not consider the regulatory and positional relationship between mRNA and lncRNA. The latter has been largely shown to possess tumor suppressive or oncogenic properties. The combined analysis of mRNA and lncRNA is likely to facilitate the identification of biomarkers with higher confidence. Results: Therefore, we have developed an lncRNA-related method to identify traditional mRNA biomarkers. First we identified mRNAs that are differentially expressed in Hepatocellular Carcinoma (HCC) by comparing cancer and matched adjacent non-tumorous liver tissues. Then, we performed mRNA-lncRNA relationship and coexpression analysis and obtained 41 lncRNA-related and -coexpressed mRNA biomarkers. Next, we performed network analysis, gene ontology analysis and pathway analysis to unravel the functional roles and molecular mechanisms of these lncRNA-related and -coexpressed mRNA biomarkers. Finally, we validated the prediction and performance of the 41 lncRNA-related and -coexpressed mRNA biomarkers using Support Vector Machine model with five-fold cross-validation in an independent HCC dataset from RNA-seq. Conclusions: Our results suggested that mRNAs expression profiles coexpressed with positionally related lncRNAs can provide important insights into early diagnosis and specific targeted gene therapy of HCC.Item Identification of novel alternative splicing biomarkers for breast cancer with LC/MS/MS and RNA-Seq(BioMed Central Ltd., 2020-12-03) Zhang, Fan; Deng, Chris K.; Wang, Mu; Deng, Bin; Barber, Robert C.; Huang, GangBackground: Alternative splicing isoforms have been reported as a new and robust class of diagnostic biomarkers. Over 95% of human genes are estimated to be alternatively spliced as a powerful means of producing functionally diverse proteins from a single gene. The emergence of next-generation sequencing technologies, especially RNA-seq, provides novel insights into large-scale detection and analysis of alternative splicing at the transcriptional level. Advances in Proteomic Technologies such as liquid chromatography coupled tandem mass spectrometry (LC-MS/MS), have shown tremendous power for the parallel characterization of large amount of proteins in biological samples. Although poor correspondence has been generally found from previous qualitative comparative analysis between proteomics and microarray data, significantly higher degrees of correlation have been observed at the level of exon. Combining protein and RNA data by searching LC-MS/MS data against a customized protein database from RNA-Seq may produce a subset of alternatively spliced protein isoform candidates that have higher confidence. Results: We developed a bioinformatics workflow to discover alternative splicing biomarkers from LC-MS/MS using RNA-Seq. First, we retrieved high confident, novel alternative splicing biomarkers from the breast cancer RNA-Seq database. Then, we translated these sequences into in silico Isoform Junction Peptides, and created a customized alternative splicing database for MS searching. Lastly, we ran the Open Mass spectrometry Search Algorithm against the customized alternative splicing database with breast cancer plasma proteome. Twenty six alternative splicing biomarker peptides with one single intron event and one exon skipping event were identified. Further interpretation of biological pathways with our Integrated Pathway Analysis Database showed that these 26 peptides are associated with Cancer, Signaling, Metabolism, Regulation, Immune System and Hemostasis pathways, which are consistent with the 256 alternative splicing biomarkers from the RNA-Seq. Conclusions: This paper presents a bioinformatics workflow for using RNA-seq data to discover novel alternative splicing biomarkers from the breast cancer proteome. As a complement to synthetic alternative splicing database technique for alternative splicing identification, this method combines the advantages of two platforms: mass spectrometry and next generation sequencing and can help identify potentially highly sample-specific alternative splicing isoform biomarkers at early-stage of cancer.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.
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