Browsing by Subject "DNA, Mitochondrial / genetics"
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Item Genetic assessment reveals no population substructure and divergent regional and sex-specific histories in the Chachapoyas from northeast Peru(PLOS, 2020-12-31) Guevara, Evelyn K.; Palo, Jukka U.; Oversti, Sanni; King, Jonathan L.; Seidel, Maria; Stoljarova, Monika; Wendt, Frank R.; Bus, Magdalena M.; Guengerich, Anna; Church, Warren B.; Guillen, Sonia; Roewer, Lutz; Budowle, Bruce; Sajantila, AnttiMany native populations in South America have been severely impacted by two relatively recent historical events, the Inca and the Spanish conquest. However decisive these disruptive events may have been, the populations and their gene pools have been shaped markedly also by the history prior to the conquests. This study focuses mainly on the Chachapoya peoples that inhabit the montane forests on the eastern slopes of the northern Peruvian Andes, but also includes three distinct neighboring populations (the Jivaro, the Huancas and the Cajamarca). By assessing mitochondrial, Y-chromosomal and autosomal diversity in the region, we explore questions that have emerged from archaeological and historical studies of the regional culture (s). These studies have shown, among others, that Chachapoyas was a crossroads for Coast-Andes-Amazon interactions since very early times. In this study, we examine the following questions: 1) was there pre-Hispanic genetic population substructure in the Chachapoyas sample? 2) did the Spanish conquest cause a more severe population decline on Chachapoyan males than on females? 3) can we detect different patterns of European gene flow in the Chachapoyas region? and, 4) did the demographic history in the Chachapoyas resemble the one from the Andean area? Despite cultural differences within the Chachapoyas region as shown by archaeological and ethnohistorical research, genetic markers show no significant evidence for past or current population substructure, although an Amazonian gene flow dynamic in the northern part of this territory is suggested. The data also indicates a bottleneck c. 25 generations ago that was more severe among males than females, as well as divergent population histories for populations in the Andean and Amazonian regions. In line with previous studies, we observe high genetic diversity in the Chachapoyas, despite the documented dramatic population declines. The diverse topography and great biodiversity of the northeastern Peruvian montane forests are potential contributing agents in shaping and maintaining the high genetic diversity in the Chachapoyas region.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 Optimization and Evaluation of qPCR Duplex Assay for mtDNA Copy Number Quantification(2020-05) Johnson, Gretchen A.; Planz, John V.; Phillips, Nicole R.; Zascavage, Roxanne R.Purpose: The mitochondrial genome (mtDNA) encodes thirteen essential proteins in oxidative phosphorylation, the cell's primary energy-generating process. Depending on the cell type and stage of development, each cell contains an average of 103 to 104 copies of mtDNA. Current methods of quantification of mtDNA copy number can be imprecise due to low efficiencies of assays and inherent imbalance of mtDNA copy number with nuclear DNA (nDNA) copy number. Accurate quantification of both mtDNA and nDNA is important when calculating the ratio of mtDNA to nDNA. The goal of this project is to optimize a duplex assay that will give precise and accurate estimates in human samples. Methods: Here we employ synthetic oligomer standards for an absolute real-time qPCR assay. The significance of using absolute qPCR is that the standard curve allows for the direct comparison of unknowns to obtain a copy number. The mitochondrial target is a site in the minor arc (MinArc), and the nuclear target is a single copy locus ([beta]2M). The accuracy of this assay was evaluated using a standard reference material (SRM2372a) and the precision was evaluated via replications. Results: This design resulted in high R2 values for the standards as well as sufficiently high efficiencies. The precision of the assay was analyzed over 6 replicated runs and was deemed effectively reproducible. The accuracy was assessed with the use of a standard reference material (SRM 2372a) and was found to be problematic [Romsos et al., 2018]. This could be from a possible dilution bias of the SRM, effectively changing the copy number ratios in a difficult to predict way [Malik et al., 2011]. An attempt to mathematically correct the data was made but did not provide any solution. Conclusion: The optimization of this assay is ongoing due to the error in accuracy. The assay has proven to be precise and reproducible with sufficient efficiency. Possible future directions include sonication of samples and SRMs to examine if dilution bias could be the cause of inaccurate SRM quantification. Other methods of possibly reducing dilution bias mentioned in Malik et al. [2011] include manual shearing and the use of DNA carriers such as tRNA. Another avenue of future research could include a different method of mathematically correcting the data post run to improve accuracy. This assay has the potential to provide data which can be used to indicate overall mitochondrial health and can be utilized in various research areas such as aging, cancer, forensics and neurodevelopment.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.Item Variation in Mitochondrial DNA Heteroplasmy from Blood, Buccal, and Hair Samples(2020-05) Colon, Natalie S.; Budowle, Bruce; Gwirtz, Patricia A.; Cihlar, Jennifer C.Mitochondrial DNA (mtDNA) sequence variation has important forensic implications. This research assesses five subjects for variation in point heteroplasmy (PHP) using blood, buccal, and head hair from five scalp sites. Five hairs from each site (25 total hairs) were sequenced for mtDNA using the Ion Chef and S5 technologies. Three hairs were used to study PHP variation along the hair shaft. Variance in hair PHP was more similar to blood (R2 = 0.3411) than buccal (R2 = 0.3059). Given sufficient signal, PHP was detected in all subjects, but was not concordant within or across all scalp sites. Along hair shafts, PHP was present in one distal hair portion. Future research is needed to elucidate PHP's impact on hair sample interpretation.