Browsing by Subject "DNA, Mitochondrial"
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Item A Continuous Statistical Phasing Framework for the Analysis of Forensic Mitochondrial DNA Mixtures(MDPI, 2021-01-20) Smart, Utpal; Cihlar, Jennifer Churchill; Mandape, Sammed N.; Muenzler, Melissa; King, Jonathan L.; Budowle, Bruce; Woerner, August E.Despite the benefits of quantitative data generated by massively parallel sequencing, resolving mitotypes from mixtures occurring in certain ratios remains challenging. In this study, a bioinformatic mixture deconvolution method centered on population-based phasing was developed and validated. The method was first tested on 270 in silico two-person mixtures varying in mixture proportions. An assortment of external reference panels containing information on haplotypic variation (from similar and different haplogroups) was leveraged to assess the effect of panel composition on phasing accuracy. Building on these simulations, mitochondrial genomes from the Human Mitochondrial DataBase were sourced to populate the panels and key parameter values were identified by deconvolving an additional 7290 in silico two-person mixtures. Finally, employing an optimized reference panel and phasing parameters, the approach was validated with in vitro two-person mixtures with differing proportions. Deconvolution was most accurate when the haplotypes in the mixture were similar to haplotypes present in the reference panel and when the mixture ratios were neither highly imbalanced nor subequal (e.g., 4:1). Overall, errors in haplotype estimation were largely bounded by the accuracy of the mixture's genotype results. The proposed framework is the first available approach that automates the reconstruction of complete individual mitotypes from mixtures, even in ratios that have traditionally been considered problematic.Item An assessment of qPCR assays for DNA concentration and degradation(2019-05) Cropper, Emily R.; Coble, Michael D.; Warren, Joseph E.; Phillips, Nicole R.Forensically challenged samples are often composed of degraded, damaged, or low template mitochondrial DNA (mtDNA). A real-time quantitative polymerase chain reaction (qPCR) assay can help determine if there is sufficient quantity and robust quality of mtDNA to move forward with downstream sequencing and analysis. The fundamental issue with qPCR is that the nominal quantity of the DNA calibrated along the commercial standard used for quantification can vary depending on the supplier and lot numbers. The National Institute of Standards and Technology (NIST) has developed a commercially available human DNA standard, Standard Reference Material (SRM) 2372a, which consists of nuclear DNA (nDNA) and mtDNA data on three wellcharacterized human genomic DNA preparations. The SRM 2372a was used to compare three qPCR assays: a non-commercial triplex assay, for mtDNA quantification, and two commercial assays, Quantifiler Trio (QFTrio) for nDNA quantification, and NovaQUANT for nDNA quantification and determination of the mtDNA/nDNA ratio. Quantification of the SRM uniformly across these three qPCR assays allowed for the conclusion that a robust, reproducible, accurate, and efficient qPCR assay is dependent on (1) the quality and reliability of the DNA standard, (2) the specificity of the qPCR chemistry, and (3) sound primers and probes, to name a few. The findings indicate that commercially available qPCR assays do not necessarily perform as marketed and should be re-verified by a validated DNA SRM.Item 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 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 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.