Browsing by Subject "mitochondrial DNA"
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Item Amplification of Mitochondrial DNA Regions HVI and HVII in its Entirety and Reducing Cycle Sequencing(2004-08-01) Ariyo, Bolanle; Joseph Warren; John Planz; Arthur EisenbergAriyo, Bolanle. Amplification of Mitochondrial DNA Regions HVI and HVII in its Entirety and Reducing Cycle Sequencing Reactions. Master of Science (Forensic Genetics), August 2004, 46 pages, 10 figures, 7 tables, 18 references. Mitochondrial DNA is widely used in the forensic community because of its high copy number in cells, location, and mode of inheritance. Yet this method of analysis is expensive, time consuming, and labor intensive, therefore labs should take steps to improve the procedure of mtDNA analysis. This study is performed to validate the use of amplifying HVI and HVII region in its entirety (2 primer sets) for use in reference samples. Amplification performed using primers F15989-R16410 (HVI) and F73-R340 (HVII). The current method of amplification is 4 primer sets at full cycle sequencing reactions. The cost of Cycle Sequencing Kit is also expensive, therefore performing half and quarter reactions would be beneficial in reducing the amount of kit consumed. To validate the use of reducing cycle sequencing reactions, half and quarter cycle reactions were performed using 2 and 4 primer sets. Results demonstrate that sequence data for reducing cycle sequence data is consistent with the sequence data using the current method. Results also show that sequence data obtained using two primer sets was consistent with sequence data amplified by the current method with the exception of two samples at length heteroplasmy polyctosine regions.Item Cell-Free mtDNA Quantification in Alzheimer's Patients from the Mexican American Population(2020-05) House, Sara R.; Phillips, Nicole R.; Hodge, Lisa M.; Zascavage, Roxanne R.Abstract Background AD is a continuous problem in the 65+ population but it is especially challenging in the Hispanic population where not only is it more prevalent but more severe than Caucasian populations. This study explores the efficacy of using peripheral blood plasma as an alternative tissue for testing as well as the usefulness for future research assisting in identifying the population structure most at risk for developing AD based upon CF-mtDNA quantity results. Materials and Methods Samples tested included a total cohort (Mexican American and Caucasian) of 177 individuals (AD=45, MCI=74, NC=58). The Mexican American subset contained 92 individuals (AD=21, MCI=53, and NC=18). Peripheral blood plasma was collected from the TARCC biobank and quantified. CF-mtDNA was then tested for significance using correlation analyses, logistic and linear regression models. Results CF-mtDNA was significantly negatively correlated with education, age, sex, and hypertensive samples in the total and Mexican American populations. The greatest difference was expected to be in CF-mtDNA quantity from NC to AD samples. Instead, the most significant difference was between MCI and NC samples. As CF-mtDNA quantity increased, the MMSE and CDRSOB scores were less impaired. Conclusion In conclusion, CF-mtDNA is an easily accessible and easily tested molecular marker of diseases that are relevant to studies for cognitive decline. Although our findings were inconsistent with current literature, they bring to light the weight of confounding factors within limited sample studies. With the completion of the full sample set associated with this study, more power is needed to overcome these issues.Item Developmental and Internal Validation of a Mitochondrial DNA Direct Amplification Kit for Forensic Reference Samples(2015-05-01) Alicea-Centeno, Alessandra; Arthur J. Eisenberg; Rhonda Roby; Dixie L. PetersEvaluation of the control region of the mitochondrial genome is a common practice for forensic casework and research purposes. Since no kit is currently commercially available for the amplification of mitochondrial DNA (mtDNA), its sequencing procedure is time-consuming and laborious. Six steps are generally followed: DNA extraction, quantification and normalization, amplification of two regions (hypervariable regions 1 and 2), cycle sequencing, capillary electrophoresis and data analysis. This project evaluated a mtDNA direct amplification kit by performing developmental and internal validations. The studies performed included sensitivity, stability, reproducibility, case- type samples, mixtures and accuracy. The mtDNA direct amplification kit successfully amplified reference samples used in each study without the need of extraction and quantification steps. In addition, mtDNA profiles were obtained from the sequenced amplification products. Using the validated direct amplification procedure in the laboratory will improve workflow, decrease operational cost and reduce the possibility of error by minimizing sample handling.Item Developmental Validation of a MPS Workflow with a PCR-Based Short Amplicon Whole Mitochondrial Genome Panel(MDPI, 2020-11-13) Cihlar, Jennifer Churchill; Amory, Christina; Lagace, Robert; Roth, Chantal; Parson, Walther; Budowle, BruceFor the adoption of massively parallel sequencing (MPS) systems by forensic laboratories, validation studies on specific workflows are needed to support the feasibility of implementation and the reliability of the data they produce. As such, the whole mitochondrial genome sequencing methodology-Precision ID mtDNA Whole Genome Panel, Ion Chef, Ion S5, and Converge-has been subjected to a variety of developmental validation studies. These validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) validation guidelines and assessed reproducibility, repeatability, accuracy, sensitivity, specificity to human DNA, and ability to analyze challenging (e.g., mixed, degraded, or low quantity) samples. Intra- and inter-run replicates produced an average maximum pairwise difference in variant frequency of 1.2%. Concordance with data generated with traditional Sanger sequencing and an orthogonal MPS platform methodology was used to assess accuracy, and generation of complete and concordant haplotypes at DNA input levels as low as 37.5 pg of nuclear DNA or 187.5 mitochondrial genome copies illustrated the sensitivity of the system. Overall, data presented herein demonstrate that highly accurate and reproducible results were generated for a variety of sample qualities and quantities, supporting the reliability of this specific whole genome mitochondrial DNA MPS system for analysis of forensic biological evidence.Item Increasing the Efficiency of Mitochondrial DNA processing of Reference Samples(2013-05-01) Morgan, Katherine N.; Rhonda RobyMitochondrial DNA (mtDNA) analysis in forensic testing is especially useful for human skeletal remains and hair samples. When a profile is generated for unidentified human remains or hair samples, comparison to a reference sample is critical to the case. The following steps are involved in the processing of family reference samples for mtDNA: DNA extraction, HV1 and HV2 amplification, cycle sequencing, electrophoresis, and analysis. Some procedures require DNA quantification and normalization. Analysis of mtDNA is expensive and time-consuming. A direct lysis and amplification method was previously shown to eliminate the need for DNA extraction, quantification, and normalization of reference samples. This study was performed to further develop, optimize, and validate its use for future implementation in routine casework of reference samples. The results have shown that quality mtDNA sequencing data can be obtained using a direct amplification method from blood and buccal samples from a variety of collection devices.Item Maternal and fetal mitochondrial gene dysregulation in hypertensive disorders of pregnancy(American Physiological Society, 2023-05-15) Ricci, Contessa A.; Reid, Danielle M.; Sun, Jie; Santillan, Donna A.; Santillan, Mark K.; Phillips, Nicole R.; Goulopoulou, StylianiMitochondrial dysfunction has been implicated in pregnancy-induced hypertension (PIH). The role of mitochondrial gene dysregulation in PIH, and consequences for maternal-fetal interactions, remain elusive. Here, we investigated mitochondrial gene expression and dysregulation in maternal and placental tissues from pregnancies with and without PIH; further, we measured circulating mitochondrial DNA (mtDNA) mutational load, an index of mtDNA integrity. Differential gene expression analysis followed by Time Course Gene Set Analysis (TcGSA) was conducted on publicly available high throughput sequencing transcriptomic data sets. Mutational load analysis was carried out on peripheral mononuclear blood cells from healthy pregnant individuals and individuals with preeclampsia. Thirty mitochondrial differentially expressed genes (mtDEGs) were detected in the maternal cell-free circulating transcriptome, whereas nine were detected in placental transcriptome from pregnancies with PIH. In PIH pregnancies, maternal mitochondrial dysregulation was associated with pathways involved in inflammation, cell death/survival, and placental development, whereas fetal mitochondrial dysregulation was associated with increased production of extracellular vesicles (EVs) at term. Mothers with preeclampsia did not exhibit a significantly different degree of mtDNA mutational load. Our findings support the involvement of maternal mitochondrial dysregulation in the pathophysiology of PIH and suggest that mitochondria may mediate maternal-fetal interactions during healthy pregnancy.NEW & NOTEWORTHY This study identifies aberrant maternal and fetal expression of mitochondrial genes in pregnancies with gestational hypertension and preeclampsia. Mitochondrial gene dysregulation may be a common etiological factor contributing to the development of de novo hypertension in pregnancy-associated hypertensive disorders.Item Next Generation Sequencing Assessment of Mitochondrial Oxidative DNA Damage in Cognitive Impairment: Shedding Light on Health Disparities in Mexican Americans(2022-08) Reid, Danielle M.; Phillips, Nicole R.; Barber, Robert C.; Sumien, Nathalie; Thorpe, Roland; Planz, John V.; Maddux, Scott D.Currently, Alzheimer's Disease (AD) is listed as the 5th and 7th leading cause of death in the US aging (individuals 65+ years of age) and general population, respectively. The US aging population has been expanding over time and is projected to triple over the next two to three decades. As this demographic shift occurs, the impact of age-related diseases, including AD will increase. Due to differences in biology, behavior, socio-economic status and health care access, this impact will not be distributed evenly across racial and ethnic divides in the US population. Unfortunately, most scientific data exists for non-Hispanic Whites (NHWs). Although limited in scope, the observations that we have for admixed populations such as Hispanics clearly show that racial/ethnic disparities and etiologies for AD exist; however, the details of these disparities remain to be elucidated. Collaborative research efforts from the Texas Alzheimer's Research and Care Consortium (TARCC) aim to identify ethnicity-specific factors that influence the development and progression of AD among Mexican Americans (MAs) compared to their NHW counterparts, and to better understand the role these factors play. Common risk factors for developing cognitive impairment (CI) in the MA population are stroke, diabetes, obesity, and depression. Although the reasons for the association between cognitive decline and comorbidities remain unclear, the incidence of these comorbid conditions is known to vary greatly across race and ethnicity. Diabetes for example is three times more prevalent among MAs relative to NHWs. Accumulating evidence indicates a correlation between common pathological changes observed in AD and DNA damage, particularly within the mitochondrial genome (mtDNA), which is positioned to be particularly vulnerable to DNA damage. Age-associated decline in mitochondrial function generates an accumulation of reactive oxygen species that are capable of damaging essential biomolecules including DNA and may help explain some of the racial and ethnic differences in etiology that exist for AD. Many forms of oxidative DNA damage exist, but oxidation of guanine (G) to 8-oxo-guanine (8oxoG) is one of the most prevalent lesions and an indicator of mitochondrial dysfunction. Damaged mtDNA, such as 8oxoG serve as important markers of age-related systemic inflammation and upon release into peripheral circulation may exacerbate the physiology and pathophysiology contributing to AD development and/or progression. Current methods for the detection of oxidized bases are limited, costly, cumbersome, and lack reproducibility. Here we describe the use of Illumina-based next-generation sequencing to quantify variants of oxidatively modified G residues in mtDNA of MA vs NHW TARCC participants. Our first study focused on investigating whether impaired mitochondrial function, represented by levels of oxidative DNA damage indicative of 8oxoG differed between MAs and NHWs. Additionally, we evaluated the effects of sex, CI, and type-2 diabetes (T2D) on risk for AD. We discovered variants representing 8oxoG from buffy coat were significantly higher in MAs compared to NHWs. Interestingly, MA females were especially affected, and years of education was significantly associated with 8oxoG load in MAs. We report suggestive evidence that 8oxoG mutational load is associated with cognitive impairment. Further, we identified individual mtDNA haplotypes that render an increased risk for oxidative DNA damage. Our second study used blood-based measurements of 8oxoG from both buffy coat and plasma to determine associations with population, sex, and T2D, for AD risk. We investigate genomic regions specially burdened by 8oxoG affecting mitochondrial function in relation to population and disease. Lastly, we characterize differences in 8oxoG mutational load between buffy coat and plasma portions of blood on assessing AD risk and endophenotype. Our results show that both buffy coat and plasma were significantly associated with population, sex, years of education, and suggest association with AD.Item Optimization of Filter Metrics for Mitochondrial DNA Sequence Analysis.(2009-08-01) Musslewhite, Pamela; Planz, John V.Quality metrics translate sequence information into numerical values which allows a software program to filter through data without human intervention. Primer specific settings for the trace score and contiguous read length in Sequence Scanner Software v1.0 (Applied Biosystems, Foster City, CA) were established using a calibration dataset of 2,817 sequence traces and validated using a second dataset of 5,617 sequence traces. Prior to optimization 51.7% of the samples required manual intervention while 28.4% require review after optimization. An evaluation of signal intensity and signal to noise ratio variables was performed and no trend was recognized for predictive modeling. Use of quality values per peak to ascertain confidence in the base call was evaluated and found to be a feasible parameter for sample quality assessment and confident base calling.Item Population Variances in the Whole Mitochondrial Genome Impacting Capture for Human Identification(2015-05-01) Skandalis, Lisa A.; Bruce Budowle; Michael Allen; Robert C. BarberThe mitochondrial genome (mtGenome) holds a wealth of information that can be used for human identification purposes. Sequencing and analyzing the entire mtGenome can help to obtain more of this information. However, library preparation steps prior to sequencing could be problematic when dealing with compromised or degraded samples. A capture/hybridization assay targeted to the entire mtGenome can enrich for the target DNA without PCR or chemically altering the DNA. The melting temperatures (Tm) of two different length fragments for an African American and Caucasian individual were mapped across the length of the genome to study any variation that might occur. While population-specific Tm differences seem to be almost negligible, there is wide intra-individual Tm variation, ranging from about 11-31°C. To overcome the issue of an assay run at a single set of temperature parameters, two separate reactions optimized for higher and lower Tm values could prove to be more efficient.Item Reduced Maternal Circulating Cell-Free Mitochondrial DNA Is Associated With the Development of Preeclampsia(American Heart Association, Inc., 2022-01-11) Cushen, Spencer C.; Ricci, Contessa A.; Bradshaw, Jessica L.; Silzer, Talisa K.; Blessing, Alexandra M.; Sun, Jie; Zhou, Zhengyang; Scroggins, Sabrina M.; Santillan, Mark K.; Santillan, Donna A.; Phillips, Nicole R.; Goulopoulou, StylianiBackground Circulating cell-free mitochondrial DNA (ccf-mtDNA) is a damage-associated molecular pattern that reflects cell stress responses and tissue damage, but little is known about ccf-mtDNA in preeclampsia. The main objectives of this study were to determine (1) absolute concentrations of ccf-mtDNA in plasma and mitochondrial DNA content in peripheral blood mononuclear cells and (2) forms of ccf-mtDNA transport in blood from women with preeclampsia and healthy controls. In addition, we sought to establish the association between aberrance in circulating DNA-related metrics, including ccf-mtDNA and DNA clearance mechanisms, and the clinical diagnosis of preeclampsia using bootstrapped penalized logistic regression. Methods and Results Absolute concentrations of ccf-mtDNA were reduced in plasma from women with preeclampsia compared with healthy controls (P0.05). While the pattern of reduced ccf-mtDNA in patients with preeclampsia remained, DNA isolation from plasma using membrane lysis buffer resulted in 1000-fold higher ccf-mtDNA concentrations in the preeclampsia group (P=0.0014) and 430-fold higher ccf-mtDNA concentrations in the control group (P<0.0001). Plasma from women with preeclampsia did not induce greater Toll-like receptor-9-induced nuclear factor kappa-light-chain enhancer of activated B cells-dependent responses in human embryonic kidney 293 cells overexpressing the human TLR-9 gene (P>0.05). Penalized regression analysis showed that women with preeclampsia were more likely to have lower concentrations of ccf-mtDNA as well as higher concentrations of nuclear DNA and DNase I compared with their matched controls. Conclusions Women with preeclampsia have aberrant circulating DNA dynamics, including reduced ccf-mtDNA concentrations and DNA clearance mechanisms, compared with gestational age-matched healthy pregnant women.