Browsing by Author "Hall, Courtney"
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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 Longitudinal microRNA profiling of neuronal-enriched exosomes associated with cognitive function and decline(2024-03-21) Subasinghe, Kumudu; Hall, Courtney; Zhou, Zhengyang; Barber, Robert; Phillips, NicoleBackground. Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that disproportionately affects several racial/ethnic groups, including Mexican Americans (MAs). Evidence suggests that early alterations in the AD brain can propagate to local and distal cells through small biological packages called exosomes. Exosomes secreted by neurons are capable of mediating cell-to-cell communication through their bioactive cargo, leading to metabolic and epigenetic reprogramming in target cells. Exosomes derived from neurons have been detected in plasma and isolated from other subpopulations using the neural cell adhesion molecule CD171. These neuronal-enriched exosomes (NEEs) cross the blood-brain barrier and thus represent an easily accessible derivative of otherwise inaccessible brain tissue in living humans. Small, non-coding RNAs called microRNAs (miRNA) are transcribed from nuclear DNA and function as strong intracellular expression regulators. miRNAs, which can be selectively packaged and transported by exosomes, have been shown to significantly alter the expression patterns of target cells. This project aims to identify the aberrant miRNA profiles that correlate with disease progression and key comorbidities (e.g., type 2 diabetes (T2D), hyperlipidemia, hypertension) in NEEs of plasma from MAs and Non-Hispanic Whites (NHWs). Hypothesis. We hypothesize that population-specific differences in NEE miRNA cargo will reflect cognitive function and decline. Methods. Longitudinal plasma samples (two time points, 2 years apart) received from the Texas Alzheimer’s Research and Care Consortium (TARCC) were processed using a two-step method that involves precipitation of total exosomes followed by NEE capture with a biotinylated antibody against the neuronal surface marker, CD171. After isolating RNA from NEEs, miRNAs were then profiled using next-generation sequencing. These profiles were then analyzed for differential miRNA expression in individuals with cognitive impairment compared to the normal control group. Results. Our preliminary quality control and sequencing data confirmed the successful isolation of miRNA from NEEs. We identified specific miRNA candidates that were differentially expressed in NEEs from cognitively impaired subjects compared to healthy controls. These miRNAs target gene networks that have been implicated in AD pathophysiology. Conclusion. This innovative workflow along with the unique sample type provides novel insight into the role of exosomal miRNA cargo in AD pathogenesis, identifying novel, population-specific targets for biomarker/diagnosis as well as therapeutic design. Further, this approach provides a conceptual framework for blood-based exosomal profiling in other complex diseases characterized by epigenetic dysregulation and systemic inflammation.Item MitoHooker: A PCR-free enrichment strategy using RNA baits for targeted detection of mitochondrial base modifications on nanopore sequencing devices(2021) Hall, Courtney; Reid, Danielle; Phillips, Nicole; Planz, JohnPurpose: Aberrant methylation and increased oxidative damage throughout the mitochondrial genome (mtDNA) have been implicated in numerous diseases ranging from cancer to neurodegeneration. Current understanding, however, is obscured by the inherent limitations of traditional detection techniques. Nanopore sequencing offers the ability to simultaneously ascertain genetic variation and base modifications without chemical treatment. While numerous copies of mtDNA are present within a sample, these sequences represent a small fraction of total genetic material competing for pore access. Therefore, this project aimed to evaluate RNA baits hybridization capture for enrichment of mtDNA prior to nanopore sequencing. Methods: Heavy and light mtDNA strands in cell-free plasma extracts were individually captured using the Arbor Biosciences myBaits Expert Mito kit. Elutant from the first capture served as input for rebaiting with the opposite probe set. Following complement synthesis by Klenow fragment, double-stranded products were multiplexed and sequenced on the MinION device. Resultant basecalled reads were mapped to the human reference genome to assess on- and off-target coverage. Base modifications in the raw data were detected using a combination of available bioinformatics tools and in-house algorithms. Results: Although overall input and throughput were significantly lower than a typical whole genome sequencing run, read count and coverage data indicate that this technique allowed mtDNA to outcompete background DNA while maintaining modified bases within the native strands. Conclusion: The workflow developed herein could provide novel insights into the complete collection of mtDNA base modifications and enable identification of disease-relevant alterations in this landscape.Item Role of DNA Methylation in Risk for Alzheimer's Disease and Type 2 Diabetes in a Mexican American Cohort(2021) Barber, Robert C.; Abraham Daniel, Ann; Hall, Courtney; Sun, Jie; Phillips, Nicole; Silzer, TalisaPURPOSE: Age related diseases such as Alzheimer's disease (AD) and type 2 diabetes (T2D) are respectively the 6th and 7th leading cause of mortality in the US. Mexican Americans, the largest ethnic minority group in the US, have an increased likelihood of developing T2D compared to their Caucasian counterparts. With the elderly Mexican American population (≥65 years old) likely to multiply 7-fold by 2050, prevalence of AD alongside T2D is predicted to increase too. Mexican Americans have an earlier onset of AD and a metabolic heavy predisposition for AD compared to Caucasians who develop inflammation-based AD. The risk for T2D and AD is multifactorial involving epigenetic factors such as methylation, which is the addition of a methyl group to the cytosine base of DNA. We aim to establish an epigenetic association between AD and T2D unique to the Mexican American population. METHODS: Participants from the Texas Alzheimer's Research and Care Consortium (TARCC), which consists of Mexican American individuals diagnosed with either AD only, T2D only or AD and T2D matched with a Caucasian counterpart were selected. Peripheral blood was drawn from participants and individual methylation profiles collected using the Illumina Infinium MethylationEPIC chip array. Differential methylation will be assessed using the Chip Analysis Methylation Pipeline (ChAMP) package in R. RESULTS: Results obtained will be analyzed using pathway and gene set enrichment analysis tools. CONCLUSIONS: Identifying possible methylation sites associated with T2D and AD unique to the Mexican American population could contribute towards developing ethnicity-specific biomarkers and treatments.Item Role of DNA Methylation in Risk for Cognitive Impairment and Type 2 Diabetes in a Mexican American Cohort(2022) Daniel, Ann Abraham; Silzer, Talisa K.; Hall, Courtney; Sun, Jie; Zhou, Zhengyang; Phillips, Nicole; Barber, Robert C.Purpose: Alzheimer's disease (AD) and type 2 diabetes (T2D) are among the leading causes of mortality among the aging Mexican American population (≥ 65 years old) in the US. This cohort is expected to be the largest aging ethnic minority group in the US by 2050. In comparison to their non-Hispanic white counterparts who are most likely to develop AD associated with inflammation, aging Mexican Americans have an earlier onset of AD and metabolism related predisposition for AD. Mild cognitive impairment (MCI) is a phenotype that often leads to AD and is also prevalent in this cohort. The presence of T2D is known to double the risk of developing MCI/AD. The risk for AD, MCI and T2D is multifactorial, involving genetics and epigenetics. Methylation is a form of epigenetic regulation whereby a methyl group is added to the cytosine base in DNA. Methylation patterns in DNA can be affected and possibly reversed by a variety of environmental factors such as lifestyle and diet. Targeting changes to methylation patterns through associated lifestyle changes could be a possible prevention method for AD, MCI and T2D in the future, particularly for minority groups affected by health disparities, such as the Mexican American population. We aim to establish an epigenetic association between cognitive impairment (identified here as AD and MCI), and T2D that is unique to the Mexican American population. Methods: For this project, 551 aging participants from the Texas Alzheimer's Research and Care Consortium (TARCC) were selected, following quality control. A cross phenotype study design will be used to assess differential methylation associated with cognitive impairment (CI) alone, T2D alone and then with both CI and T2D simultaneously. For the first stage of this project, 299 Mexican American and 252 non-Hispanic white participants were stratified into groups of individuals diagnosed with CI alone and controls without CI within each ethnic group. In the second stage, this cohort will be stratified into individuals with T2D alone and controls without T2D. The third stage will stratify participants into those with both CI and T2D versus normal healthy controls. Lastly, any differential methylation associated with each ethnic group will be compared and contrasted. Peripheral blood drawn from participants was used to obtain individual methylation profiles using the Illumina Infinium MethylationEPIC chip array. Differential methylation will be assessed using the Chip Analysis Methylation Pipeline (ChAMP), limma and cate packages in R. The Beta MIxture Quantile dilation (BMIQ) method will be used for data normalization. Results: Gene set enrichment and pathway analysis tools will be used to analyze results. Conclusions: Identifying methylation sites associated with CI and T2D could contribute towards developing biomarkers that are ethnicity-specific for the Mexican American population and possibly lead towards more effective medical treatment in the future.Item Role of methylation in risk for cognitive impairment in Mexican Americans(2023) Abraham Daniel, Ann; Silzer, Talisa; Sun, Jie; Zhou, Zhengyang; Hall, Courtney; Phillips, Nicole R.; Barber, Robert C.Purpose: Mexican Americans (MAs) are the largest aging (›65 years old) and growing US ethnic minority group, with a unique risk for cognitive impairment (CI) in comparison to non-Hispanic whites (NHWs). MAs have an earlier age of onset and a risk for CI that is largely metabolism related in contrast to NHWs who have a more inflammation-based risk for CI. CI is defined in this study as individuals diagnosed with either Alzheimer’s disease (AD), or mild cognitive impairment (MCI) (a likely precursor to AD). Risk for CI is multifactorial and involves an epigenetic form of gene regulation called DNA methylation, which involves the addition of a methyl group to the cytosine base of DNA. DNA methylation patterns can be altered or possibly reversed through changes in environmental factors such as diet and lifestyle. Our aim was to identify differentially methylated sites of the genome associated with CI and determine DNA methylation profiles that are specific to MAs and NHWs. Methods: Peripheral blood was drawn from 551 Texas Alzheimer's Research and Care Consortium participants (299 MAs and 252 NHWs) and DNA was typed on the Illumina Infinium MethylationEPIC chip array, assessing >850,000 CpG genomic sites. Participants were compared according to cognitive status (control versus CI(AD/MCI)) among each ethnic group. Beta values that represent relative degree of methylation were normalized using the Beta MIxture Quantile dilation (BMIQ) method. Differential methylation between control and CI was assessed using the Chip Analysis Methylation Pipeline (ChAMP), limma and cate packages within R studio. Results: Two significant differentially methylated sites were associated with CI at an FDR-adjusted p-value threshold <0.05: cg13135255 in MAs and cg27002303 in NHWs. Three differentially methylated sites were suggestively associated with CI at an FDR-adjusted p-value threshold <0.1: cg01887506 and cg10607142 in MAs, and cg13529380 in NHWs. Four of the five significant and suggestively differentially methylated sites were hypermethylated in CI compared to normal controls, except for hypomethylated site cg13529380. The site most significantly associated with CI was cg13135255 within the CREBBP gene in MAs (FDR-adjusted p-value = 0.029). The CREBBP protein is a histone acetyltransferase, involved in epigenetic regulation, and plays a role in memory formation. Conclusion: This is the first study to report these specific CpG sites as either significantly or suggestively associated with CI among MAs and NHWs. These sites may be used in addition to other methylated sites to develop risk assessments that are ethnicity specific for CI. Following further validation and replication in other cohorts these sites may aid development of ethnic specific therapeutics that could deter or delay CI in the future.Item Sequencing Long Amplicon Microsatellite Loci Using the Oxford Nanopore Technologies MinION Device(2019-03-05) Zascavage, Roxanne R.; Sedlazeck, Fritz; Planz, John V.; Hall, CourtneyPurpose: Forensic DNA typing exploits the high variability of short tandem repeat (STR) markers to differentiate individuals. Typical STR workflow consists of PCR followed by separation and detection via capillary electrophoresis (CE). Despite the power and reliability of current techniques, variations in nucleotide sequences are masked in size-based DNA profiles. Nanopore sequencing has the ability to provide long-read DNA sequence data that allows for accurate alignment and identification of single nucleotide polymorphisms (SNPs) both within and around STRs of interest. Detection of hidden sequence variation significantly expands the resolving power of STRs and aids in interpretation of more challenging samples. This project aimed to evaluate the applicability of nanopore sequencing to forensically-relevant autosomal and Y STR markers. Methods: Twenty unrelated individuals, two control DNAs, and three NIST-traceable standards were evaluated for 45 STR loci. Primer sets targeting 800 base pair amplicons containing the repeat and flanking regions were designed and multiplexed. Amplicons from each sample were barcoded and sequenced on the ONT MinION device using 1D read chemistry and SpotON flow cells (vR9.4.1). Raw reads were basecalled with MinKNOW’s real-time, local base caller. Sequence data were separated by barcode, merged by sample, and mapped to the human reference sequence (GRCh37/hg19) using NextGenMap-LR (NGMLR). Variations in motif composition and flanking SNPs were detected using Sniffles and visualized with Integrative Genomics Viewer (IGV). Consensus sequences and variant reports were compiled for each sample. Size-based allelic designations were predicted and compared to those generated via CE to evaluate concordance between the STR typing approaches. Results: High quality sequencing results were obtained for all STR loci interrogated. Concordance between size-based allelic designations revealed the reliability of nanopore sequencing data analyzed using this customized pipeline. Identification of flanking SNPs within the longer amplicons added variations that could differentiate alleles with the same motif structure, enriching discrimination potential. Conclusions: Complete nucleotide sequence data for repeat and flanking regions enhances the resolving power over that of current STR typing techniques. This project sets the foundation for future development of STRs for biomedically-relevant regions and potential forensic applications.Item Taking the bait: A PCR-free enrichment strategy for nanopore sequencing applications(2020) Planz, John V.; Sedlazeck, Fritz; Zascavage, Roxanne R.; Hall, CourtneyPurpose: DNA methylation is a critical epigenetic modification involved in regulating gene expression. Although aberrant cytosine methylation has been implicated in diseases ranging from neurological disorders to cancer, current understanding is obscured by the inherent limitations of bisulfite conversion. Nanopore sequencing offers the ability to simultaneously ascertain genetic variation and base modifications without chemical treatment. While numerous copies of target regions may be present within a sample, this represents a small fraction of total genetic material competing for pore access. Here we evaluate RNA bait hybridization capture for enrichment of mitochondrial DNA (mtDNA) prior to nanopore sequencing. Methods: Heavy and light strands were individually captured using the Arbor Biosciences myBaits Mito panel. Elutant from the first capture served as rebaiting input with the opposite probe set. Double-stranded products subsequently generated were multiplexed and sequenced on the MinION device. Resultant data were separated by barcode and mapped to the human reference genome. Read counts were normalized for coverage comparisons and methylation was detected using Tombo. Results: Overall input and throughput were significantly lower than a typical whole genome sequencing run, however mtDNA read counts indicate successful enrichment. In addition to ensuring target regions outcompete background DNA, these techniques allowed for methylation detection within native strands. Conclusion: The significance of a PCR-free enrichment strategy for nanopore sequencing applications extends beyond mtDNA. These techniques have been used to capture challenging regions within the rat genome and could provide novel insights into the genetic and epigenetic landscape of other biomedically relevant regions.Item Taking the Bait: Utilization of Probe-Capture Enrichment in Human Remains Identification(2024-03-21) McBroom, Katherine; Kesharwani, Rupesh; Kapema, Bupe; Hall, Courtney; Phillips, Nicole; Sedlazeck, Fritz; Zascavage, Roxanne R.Purpose: Human remains are frequently encountered in forensic laboratories, coming from crime scenes, mass graves, historical samples, mass disasters, and military conflicts. Short tandem repeat (STR) markers evaluated via capillary electrophoresis (CE) are the gold standard for human remains identification (HRID) in forensic investigations due to their high variability and robust database of comparative samples. However, CE excludes valuable sequence-level information both within and around STRs and is often unsuccessful when used on challenged and degraded samples. The problem forensic laboratories face is choosing between depleting sample volumes to repeat individualizing STR analysis or perform costly, time-consuming, and less discriminatory mitochondrial DNA analysis. New DNA sequencing methodologies combined with novel enrichment techniques may provide a more effective platform that overcomes the most common challenges associated with HRID. Our goal for this portion of the project was to design a RNA-baiting assay to capture forensically relevant STRs. Methods: A custom myBaits panel targeting forensically relevant regions of interest was designed with Arbor Biosciences. DNA extract from whole blood samples was used. Samples were barcoded with Illumina-compatible barcode adapters, pooled, and then probe-captured, resulting in single stranded DNA covering our targeted regions. Fill-in PCR was performed on the captured single stranded DNA using Illumina primers to create DS DNA, which was then sequenced on the MiSeq. Our data was analyzed using our custom script, STRspy, which uses minimap 2 or BWA-mem to align the data to the designated reference database. STRspy then evaluates the reads aligning to each designated loci (forensically relevant STRs in this case), classifying the allele calls as well as the SNPs in the flanking region to create sequence-based genetic profiles. These calls also include the length-based allele designation for each sequence-based allele, therefore allowing the results to be compatible with current CODIS data. Results: We found that our RNA-baiting assay was successful in capturing our targeted regions of interest. The STR regions targeted for enrichment showed significantly enhanced coverage compared to the coverage across the entire genome. Conclusions: Our results indicate that probe-capture is a viable enrichment technique for DNA samples.