Browsing by Subject "genetics"

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Associations of social and genetic factors with sarcoma incidence and outcomes.
(2020-08) Diessner, Brandon
The risk factors for the development or progression of sarcomas remain mostly obscure. This is largely due to the extreme rarity of the disease, oftentimes constraining investigators to evaluate histologically distinct sarcoma subtypes as a single group or else risk analyses of insufficiently low statistical power. In this dissertation, we aimed to evaluate the genetic and non-genetic risk factors for the development or progression of individually rare subtypes of sarcoma. The first project utilized the SEER Census Tract-level Socioeconomic Database to assess the associations of census tract-level socioeconomic status (CT-SES) or race/ethnicity on the incidence rates or odds of metastasis at diagnosis for sarcoma subtypes diagnosed across the age span. Overall, we found race/ethnicity to be more often associated with sarcoma incidence than CT-SES, particularly in younger age groups. Additionally, in adults, we found SES-related factors increased the odds of metastasis at diagnosis for several soft tissue sarcomas, but not bone sarcomas. In Project 2, we aimed to describe the contribution of common genetic variation to osteosarcoma (OS) development by leveraging a newly created dataset of open chromatin regions (OCR) of osteoblasts. We found an enrichment of OS-associated loci in these regions, indicating that several common variants contribute a weak or moderate effect on osteosarcoma development by altering regulatory mechanisms that localize to an osteoblast’s OCR. Lastly, we sought to determine the associations of risk alleles discovered by GWAS in Europeans and genetic ancestry on Ewing sarcoma (ES) risk in Latinos. We found the effect of these alleles generalize to Latinos. Additionally, we report a residual inverse association between African genomic ancestry and ES risk. These data indicate that other ancestry-specific genetic variants may influence ES susceptibility and explain the observed racial disparities. Overall, our findings add to the scientific knowledge of an exceedingly understudied group of cancers.
Chimeric Criminals
(2013-02-12) Kaye, David H.
According to some commentators, an obscure genetic condition known as chimerism “could undermine the very basis of the forensic DNA system” and force a reconsideration of “the entire project of forensic DNA.” This conclusion is as unfounded as it is unnerving. Chimerism is a consideration in, but not a real obstacle to DNA identification. This essay explains why.
The Enigma of Genetic Linkage in Molecular Breeding for Maize
(2017-06) Sleper, Joshua
Linkage among quantitative trait loci prevents the release of hidden genetic variation, but also preserves desirable gene combinations. This dissertation, which includes three studies, shows the continuing enigma of linkage in maize (Zea mays L.) breeding. The first study aimed to determine if the additional recombinations in doubled haploids induced from F2 instead of F1 plants leads to a larger genetic variance and a superior mean of the best lines. In two maize populations, inducing doubled haploids from F2 plants did not improve the mean, and it increased the genetic variance for moisture, but not for yield and plant height. The second study aimed to determine if multi-allelic markers or haplotypes improve the prediction accuracy of genomewide selection in three-way breeding populations, which could have three alleles per locus. In both simulated and empirical maize populations, accounting for multiple alleles did not improve the prediction accuracy over a biallelic model. The third study aimed to determine if genomewide markers can be used to partition trait effects into independent and correlated portions, and if selection on the independent portion was more effective than selection on the entire trait. Results from four cycles of selection showed that selection only for the independent portion did not lead to higher responses for yield, moisture, and plant height. Overall, genetic linkage both assists and confounds molecular breeding efforts in maize.
Epigenetic regulatory mechanisms that govern cardiovascular development
(2022-06) Sierra Pagan, Javier
Cardiovascular disease (CVD) remains the number one cause of death in the United States and the World. The clinical outcomes of patients with heart failure, a form of CVD, remain poor because current clinical therapies do not address a critical feature of heart failure which is the loss of functional cardiac muscle. To decrease the morbidity and mortality in these patients, several strategies are being developed to replace the loss of functional cardiac muscle with new one. Two attractive strategies for treating CVD involve converting cardiac fibroblasts (reprogramming) into functional muscle or vascular cells and promoting cell cycle re-entry of adult cardiomyocytes following cardiac injury to replace the dead muscle. While the adult mammalian heart has limited regenerative potential following injury, the embryonic and neonatal mammalian heart has a remarkable regenerative capacity. Therefore, our goal for these studies was to define new factors and mechanisms that could enhance repair and regeneration in the adult mammalian heart. To this end, in this thesis, we identified novel epigenetic regulatory mechanisms that govern cardiovascular development, particularly within the vascular and cardiac muscle lineages. Our first finding is that we identified that ETV2 functions as a pioneer transcription factor that relaxes closed chromatin and regulates endothelial development. We did this by comparing engineered embryonic stem cell (mESCs) differentiation and reprogramming models (MEFs) with multi-omics techniques, we demonstrated that ETV2 was able to bind nucleosomal DNA and recruit BRG1. The recruitment of BRG1 led to the remodeling of chromatin around endothelial genes and helped to maintain an open configuration, resulting in increased H3K27ac deposition. Our second finding is that we discovered a signaling cascade where ETV2 regulates RHOJ expression during endothelial progenitor cell migration. We did this by combining computational genomics (RNAseq, ATACseq and ChIPseq) to discover that ETV2 regulates migratory pathways through the expression of RHOJ, particularly in developing endothelial progenitor cells (E7.75 and E8.5 mouse embryos and developing mESCs). Our third finding is that we identified FOXK1 as an essential transcriptional and epigenetic regulator of cardiovascular development. We used mESCs that lacked FOXK1 expression and discovered that in its absence, cardiac muscle development is significantly affected, both at the transcriptional and chromatin level. Mechanistically, we also discovered that FOXK1 represses Wnt signaling, particularly Wnt6, to promote the development of cardiac progenitor cells. ETV2 has the capacity to reprogram fibroblasts to mature vascular cells and our findings identified new mechanisms we can explore to better reprogram cardiac fibroblasts. Additionally, FOXK1 is a known cell cycle regulator and together with this newly identified role in the cardiovascular system, it becomes an attractive molecule that could be used to promote cell cycle re-entry of adult cardiomyocytes following ischemic injury. Altogether these studies provide exciting data for the field of cardiac regeneration but future studies will be needed in vivo to determine the potential benefit of these molecules following cardiac injury.
Evaluating the ability of microsatellite DNA markers and otolith microchemistry to distinguish spatially separated populations and identify recruitment sites of common carp (Cyprinus carpio) in interconnected lake systems of the North American Midwest
(2017-10) Swanson, Reid
The common carp (Cyprinus carpio) is an invasive fish whose populations have grown to ecologically damaging levels in the North American Midwest and many areas throughout the world. Recent research has shown that abundance of this species in areas of the North American Midwest is driven by its propensity to use shallow basins as productive nursery habitats. The ability of managers to discriminate which shallow basins are producing carp across a large sub-watershed of interconnected lakes has the potential to increase the efficacy of management practices which are aimed at the disruption of successful recruitment (i.e. surviving to join adult population). This study assessed whether carp nurseries could be distinguished based on differences in 12 microsatellite DNA markers in carp across the twin cities metropolitan area (n=1023) and the concentrations of 11 trace elements measured in carp otoliths collected in the Six-Mile Creek sub-watershed, Minnesota, USA (n=157). I found that genetic assessment could separate carp populations at a regional scale, but not between individual putative nurseries. Microchemical otolith analysis revealed that it is feasible to classify carp to their capture locations, discriminate between nursery and non-nursery habitat types, and distinguish juvenile carp from individual nursery sites. Elemental signatures of otoliths were obtained from laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Ratios of aluminum (Al), barium (Ba), copper (Cu), iron (Fe), potassium (K), lithium (Li), magnesium (Mg), sodium (Na), phosphorus (P), and strontium (Sr) to calcium (Ca) in otolith edges differed significantly among carp from all eight capture sites in a 70 km2 watershed. Ratios of Ba, Fe, Li, manganese (Mn), and P to Ca differed significantly among juvenile carp from three nursery basins. Ratios of Al, Ba, Cu, Fe, Li, K, Na, P, and Sr to Ca were significantly different between nursery and non-nursery habitat types. Quadratic discriminant analysis (QDA) could accurately classify otoliths to collection site (total accuracy 54%). QDA had increased accuracy when restricted to juvenile carp (76%) and classifying carp to nursery and non-nursery habitat types (87%) in contrast to individual sites. Further evaluation of differences between elemental signatures of the core and edge region (i.e. recent and natal signatures) and water samples from multiple years suggests that elemental parameters in otoliths and water are changing across time preventing identification of past recruitment sources. Identification of past natal origins of carp in these systems using otolith microchemistry will require obtaining continuous signatures across larger spatial and temporal scales.
Genetic risk factors of pediatric germ cell tumors and the late effects of their treatment
(2021-09) Hubbard, Aubrey
Pediatric germ cell tumors (GCTs) are a heterogeneous group of tumors and their etiology is poorly understood. There are distinct differences between GCTs that arise in early childhood and those in adolescents and young adults, but recent studies of GCTs implicate genetics in the etiology of disease across all age groups. In Manuscript 1, we interrogated the genetically regulated expression of genes within the cell cycle control or apoptosis cellular pathways for their role in pediatric GCT risk. Our results suggest a previously identified association with an intronic variant near BAK1, a pro-apoptotic gene, may be acting through the reduced expression of BAK1. Hispanic children have the highest risk of pediatric GCTs overall, although incidence rates by race/ethnicity do vary by tumor location, sex, and age at diagnosis. No risk factors have been identified to date to explain the excess risk in Hispanic children and in countries in Central America. In Manuscript 2, we employed admixture mapping methods to interrogate genomic regions specific to Hispanic admixture to explore genetic loci contributing to the excess risk in GCTs among Hispanic children. We did not find any genetic loci that reached our threshold for statistical significance, but Amerindian ancestry regions on chromosome 7 were overrepresented among our top results and may warrant further exploration in a sample greater powered for subgroup analyses. Pediatric GCT survival has approached 90% due to the success of platinum-based therapies, but there are substantial late effects of treatment. Little is known about the burden of late effects in pediatric GCT survivors, as they were excluded from the Childhood Cancer Survivor Study. However, pediatric GCT survivors comprise the third largest group of pediatric cancer survivors in the United States. Platinum-based chemotherapy is a risk factor for chemotherapy-induced peripheral neuropathy (CIPN), which is a progressive, debilitating condition with significant effects on a survivor’s quality of life. In Manuscript 3, we characterized the prevalence of CIPN in pediatric GCT survivors and explored the genetic susceptibility to CIPN in survivors that received platinum-based chemotherapy. We found that 24% of our pediatric GCT survivors had at least two symptoms of CIPN after an average of 9 years following their diagnosis. Our genetic studies did not identify genetic risk factors although power was limited to detect variants that reached genome-wide significance. Overall, our first project provided information related to the functional significance of a previously identified variant, our second manuscript is the first genetic study in any age group to include cases of non-European ancestry and our final manuscript is the first study of CIPN in the pediatric age group.
Genetic Variants Associated with Tacrolimus Metabolism in Kidney Transplant Recipients
(2023-08) Dorr, Casey
This master’s thesis focuses on the work completed during my K01 award Genetic Variants Associated with Tacrolimus (TAC) metabolism in Kidney Transplant Recipients. In Chapter 1, I discuss the significance and innovations of this project. In Chapter 2, I present reformatted manuscript published in the Pharmacogenomics Journal titled: Identification of genetic variants associated with tacrolimus metabolism in kidney transplant recipients by extreme phenotype sampling and next generation sequencing. In Chapter 3, I present a reformatted manuscript published in Drug Metabolism and Disposition titled: CRISPR/Cas9 genetic modification of CYP3A5 *3 in human hepatocytes leads to cell lines with increased midazolam and tacrolimus metabolism. Chapter 4 is the general conclusions, future directions and take away messages.
The Genetics of General Cognitive Ability
(2013-05) Kirkpatrick, Robert
General cognitive ability (GCA) is a highly heritable trait, with correlates in numerous other domains. This dissertation reports the results of three studies of the genetics of GCA, conducted with participants from the Minnesota Center for Twin & Family Research (MCTFR). Study #1 (N = 7,100) is a genome-wide association study plus other analyses that exploit genome-wide single-nucleotide polymorphism (SNP) data. Study #2 (N = 6,439) is an association study of a different class of genetic polymorphism, the copy-number variant (CNV). In this study, we detect CNVs from genome-wide SNP-allele-probe intensity data. We aggregate them into genome-wide mutational burden scores and also carry out genome-wide association scans for specific CNVs. Study #3 is a biometric moderation study in a sample of 2,494 pairs of twins, full siblings, and adoptive siblings. We compared models by their sample-size-corrected AIC, and based our parametric inference on model-averaged point estimates and standard errors. Taken as a whole, these three studies demonstrate that GCA is substantially heritable and massively polygenic, but it is also influenced by environmental factors, and its heritability can be moderated by contextual variables like age and family-of-origin socioeconomic status (SES).
Identification of genetic loci underlying equine metabolic syndrome and laminitis risk
(2019-10) Norton, Elaine
Laminitis is a painful, debilitating disease of the hoof, often resulting in these horses being humanely euthanized due to uncontrolled pain. The most commonly cited cause of this life-threatening disease is a clustering of clinical signs resulting from a metabolically efficient phenotype, termed equine metabolic syndrome (EMS). While EMS is a commonly diagnosed syndrome, knowledge of the underlining pathophysiology is lacking and recommendations for diagnostic criteria are vague and inconsistent. EMS is thought to be complex disease, and identification of its underlying genetic risk factors and key gene-by-environment interactions will improve our understanding of EMS pathophysiology and allow for early detection of high-risk individuals and intervention prior to the onset of laminitis. We hypothesized that major genetic risk factors leading to EMS and laminitis susceptibility are shared across breeds of horses, and that differences in the severity and secondary features of the EMS phenotype between breeds, or between individuals within a breed, are the result of modifying genetic risk alleles with variable frequencies between breeds. To test these hypotheses, my PhD thesis has consisted of using phenotype and genotype data on 286 Morgan horses and 264 Welsh ponies, two high risk breeds for EMS. Phenotype data collected on all horses included: signalment, medical history, laminitis status, environmental management (feed, supplements, turnout and exercise regimen), and morphometric measurements (body condition score (BCS), wither height, and neck and girth circumference). After an eight hour fast, an oral sugar test (OST) was performed using 0.15mg/kg Karo lite corn syrup. Biochemical measurements included baseline insulin, glucose, non-esterified fatty acids (NEFA), triglycerides (TG), adiponectin, leptin and ACTH; and measurements 75 minutes after the OST included insulin (INS-OST) and glucose (GLU-OST). For inclusion in the study, each farm had to have at least one control and one horse with clinical signs consistent with EMS under the same management. Single nucleotide polymorphism (SNP) genotyping was performed on all horses. Haplotype phasing and genotype imputation up to two million SNPs was performed on horses genotyped on lower density arrays using Beagle software. Quality control on the imputed data was performed using the Plink software package. After genotype pruning, 1,428,337 and 1,158,831 SNPs remained for subsequent analysis in the Welsh ponies and Morgan horses, respectively. In chapter 2, SNP genotype data from the Welsh ponies and Morgan horses were used to estimate the heritability of the nine EMS biochemical measurements. Heritability (h2SNP) was estimated using a restricted maximum likelihood statistic with the inclusion of genetic relationship matrix, which was corrected for linkage disequilibrium (regions of the genome which are not independent as they are inherited together). The confounders of age, sex and season were included in the model based on the Akaike information criteria. In the Welsh ponies, seven of the nine biochemical traits had h2SNP estimates with p-values that exceeded the Holm-Bonferroni corrected cut-off as follows: triglycerides (0.31), glucose (0.41), NEFA (0.43), INS-OST (0.44), adiponectin (0.49), leptin (0.55), and insulin (0.81). Six of the nine EMS traits in the Morgans had h2SNP estimates with p-values that exceeded the Holm-Bonferroni cutoff as follows: INS-OST (0.36), leptin (0.49), GLU-OST (0.57), insulin (0.59), NEFA (0.68), and adiponectin (0.91). Insufficient population size and high trait variability may have limited power to obtain statistically significant h2SNP estimates for ACTH (both breeds), glucose and triglycerides in Morgans and GLU-OST in Welsh ponies. These data provide the first concrete evidence of a genetic contribution to key phenotypes associated with EMS and demonstrate that continued research for identification of the genetic risk factors for EMS phenotypes within and across breeds is warranted. Although heritability estimates provide valuable insight on the genetic contribution to a trait, they do not provide information on the number of contributing genes, specific genes involved, or where these genes are located within the genome. Genome wide association analyses (GWA) use SNP genotype data to identify those key regions of the genome that are associated with a trait. The objectives of chapter 3 were to (i) perform within breed GWA to identify significant contributing loci in Welsh ponies and Morgans, and (ii) use a meta-analysis approach to identify shared and unique loci between both breeds. For each trait, within breed GWA were performed from the imputed SNP genotype data using custom code for an improved mixed linear regression analysis. Prior to analysis, traits were adjusted to account for known covariates, with sex and age included as fixed effects and farm as a random effect. GWA meta-analysis was performed with a random effects model using the Morgans and Welsh pony GWA summary data from the 688,471 SNPs that were shared between breeds. To define the boundaries of the region, a pairwise comparison of linkage disequilibrium (LD) was calculated for all SNPs within the region. A custom code was used to identify regions where LD for all SNPs dropped below the LD threshold of 0.3 and spanned at least 100kb both 5' and 3' to the widest peak of LD within the window, which was used to define the boundaries of the ROI. An LD-region was identified as shared if it was within the boundaries of another LD-region and prioritized as described above for the fixed regions. Regions were prioritized based on whether they were identified as shared between breeds on meta-analysis (high priority), shared across traits (medium priority), or found in a single breed but not shared across traits (low priority). Prioritization resulted in 56 high, 26 medium, and 7 low priority genomic regions for a total of 1853 candidate genes in the Welsh ponies, and 39 high, 8 medium and 9 low priority regions for a total of 1167 candidate genes in the Morgan horses. Meta-analysis identified 65 of these regions that were shared across breeds. These data demonstrate that EMS is a polygenic trait with both across breed and breed specific genetic variants. In chapter 4, we utilized imputed whole-genome sequencing (WGS) and linear regression analysis in order to fine-map selected high priority LD-ROI in both the Morgan horses and Welsh ponies. LD-ROI were fine-mapped if they contained at least 5 SNPs with one SNP exceeding the threshold for genome-wide significance. Five fine-mapped regions from each breed were further interrogated for predicted impact using variant annotation. Protein-coding genes containing non-coding or coding variants within the fine-mapping region were then further prioritized based on known function and biological evidence in other species utilizing the PubMed search engine. A total of 19 positional candidate genes were identified as having biological evidence for a role in EMS. These data provide support for the process of fine-mapping GWA ROI by increasing marker density and using biological evidence across species to further prioritize candidate genes. In chapter 5, a missense mutation in the first exon of HMGA2 was identified as a putative functional allele for height and EMS phenotypes in Welsh ponies. It is well recognized that ponies (short horses) are at high risk for developing EMS; and in humans shorter individuals have an increased risk of developing cardiovascular disease, type II diabetes and metabolic syndrome. We hypothesized that genetic loci affecting height in ponies have pleiotropic effects on metabolic pathways and increase the susceptibility to EMS. Pearson’s correlation coefficient identified an inverse relationship between height and baseline insulin (-.26) in the Welsh ponies. Genomic signature of selection analysis was performed using a di statistic and identified a ~1.3 megabase region on chromosome 6, that was also identified on GWA. Haplotype analysis using HapQTL confirmed that there was a shared ancestral haplotype between height and insulin. This region contributed ~40% of the heritability for height and ~20% of the heritability for insulin. HMGA2 was identified as a candidate gene, and sequencing identified a single a c.83G>A variant (p.G28E) in HMGA2, previously described in other small stature horse breeds. In our cohort of ponies, the A allele had a frequency of .76, was strongly correlated with height (-.75) and was low to moderately correlated with metabolic traits including: insulin (.32), insulin after an oral sugar test (.25), non-esterified fatty acids (.19) and triglyceride (.22) concentrations. For this allele, model analysis suggested an additive mode of inheritance with height and a recessive mode of inheritance with the metabolic traits. This was the first gene identified as having a pleotropic effect for EMS. In conclusion, the results of my thesis are major steps forward in understanding the genetic contributions of EMS in two high risk breeds. Future directions include the continued identification of the specific genes and alleles contributing to EMS and could include prioritization of the positional candidate genes identified in aim 2 via (1) identification of biological candidate genes based on known gene function and evidence from other species; (2) use of whole genome sequencing and linear regression analysis to fine map regions; (3) use of intermediate phenotypes such as metabolomics or transcriptomics to identify shared regions; or (4) network analysis for identification of genes within similar, relevant pathways.
Immune-Mediated Myositis in Horses: From phenotype to genotype
(2016-05) Durward-Akhurst, Sian
Background: Equine immune-mediated myositis (IMM) is a painful and debilitating condition of predominantly Quarter Horse (QH) and related breeds. The epaxial and gluteal muscles are most severely affected and muscle atrophy can be dramatic, with 50% of the affected muscle mass being lost in <72 hours. Diagnosis is based on a muscle biopsy of affected muscles and the identification of lymphocytes invading myofibers and in some cases surrounding blood vessels. The pathophysiology is presumed to be immune-mediated, but further evidence is needed to confirm this. Abnormal expression of major histocompatibility Complex (MHC) has been identified on muscle fibers from most human IMMs and provides the most consistent indication of an immune-mediated mechanism. The restriction of IMM to primarily QH and related breeds, particularly in certain bloodlines suggests that there is a genetic susceptibility underlying IMM. Hypothesis: Quarter Horses are genetically susceptible to an immune mediated myositis that is characterized by abnormal expression of MHC class I and/or class II on the sarcolemma of myofibers. Specific Aim 1: To determine if abnormal MHC class I and II expression is present on the sarcolemma of myofibers of horses with active IMM in the presence or absence of myofiber lymphocytic infiltrates. Specific Aim 2: To characterize the subtypes of lymphocytes in the myofibers of horses with active IMM and correlate this with MHC expression. Methods: Immunohistochemical staining for MHC I, II, CD4+, CD8+, CD20+ lymphocytes was performed on archived muscle samples of IMM (21 horses) and controls (3 healthy and 6 disease controls). Scores were given for MHC I and II and for lymphocytic subtypes. Results: A degree of sarcolemmal MHC I and II expression was present in 81% and 71% of IMM horses, respectively. CD4+, CD8+, and CD20+ cells were present in 20/21 IMM horses with a CD4+ predominance in 48% of cases. MHC I score was positively correlated with MHC II (r = 0.89, p = <0.001) and CD8+ (r= 0.64, p = 0.002) and CD20+ (r = 0.66, p = 0.001) lymphocyte and macrophage scores (r = 0.70, p = <0.001). MHC II scores were positively correlated to CD8+ (r = 0.59, p = 0.005), CD20+ (r = 0.61, p = 0.004) lymphocyte and macrophage (r = 0.70, p = <0.001) scores. Specific Aim 3: To determine if equine IMM is significantly associated with a region of the equine genome using a genome-wide association (GWA) study. Methods: DNA was extracted from blood and muscle of 36 IMM horses and 54 healthy controls of QH-related breeds that were housed in similar environments. Sequencing was performed on equine 50K and 70K single nucleotide polymorphism (SNP) arrays. A GWA was performed across 40,811 SNPs that passed quality control. To account for elevated genomic inflation, statistical analysis was performed using GEMMA and GRAMMAR-GC software. Results: A significant association was identified between IMM and a 2 MB region on equine chromosome 11. Five SNPs in 3 haplotype blocks reached genome-wide significance using the 2 different statistical methods to account for population stratification. The significant region contains 6 myosin heavy chain genes expressed in skeletal muscle, including MYH2, which has been associated with a human IMM. Conclusions: Equine IMM is characterized by MHC I and II expression on the sarcolemma of myofibers during an acute CD4+ and CD8+ lymphocytic inflammatory episode. There is an approximately 2MB region on equine chromosome 11 that is associated with the development of the disease. Sequencing of the MYH genes in IMM cases and unaffected controls is warranted to identify variants that cause IMM in Quarter Horses.
Integrating Genomics and Metabolomics to Inform Breeding for Powdery Mildew Resistance in Grapevine
(2018-08) Teh, Soon Li
Two powdery mildew resistance loci have been identified using pedigree-connected F1 mapping families at the University of Minnesota grape breeding program. A consensus linkage map of the resistant parent (MN1264) was developed for genetic mapping. The resistance loci were mapped on chromosomes 2 and 15, with additive effects accounting for over 30% phenotypic variation. Marker haplotypes, hap+chr2 and hap+chr15, were constructed to trace the inheritance of resistance loci in grandparent-parent-progeny relationships. Both hap+chr2 and hap+chr15 in the resistant F1 progeny were inherited from parent MN1264, that originated from grandparent ‘Seyval blanc’. Additionally, two microsatellites markers (i.e., UDV-015b and VViv67) were identified to be associated with hap+chr15, and can be applied for marker-assisted selection. In a follow-up study to characterize metabolic changes attributed to hap+chr2 and hap+chr15, a metabolomic experiment was conducted on whole-plant propagated grapes in a time-course response to in vivo inoculation. The use of several multivariate analyses systematically identified 52 biomarkers that were associated with hap+chr2, and 12 biomarkers with hap+chr15. In a temporal assessment of biomarkers, the discriminating metabolic changes distinguishing resistant and susceptible individuals appeared to be occurring from 24 to 48 hours after inoculation.
Metabolic and Genetic Determinants of Urolithiasis in a Natural Canine Model
(2014-06) Furrow, Eva
Calcium oxalate (CaOx) kidney stones are a common health problem across the world. There is a substantial inherited component to CaOx stone risk, but susceptibility genes have largely evaded identification. The goal of this thesis was to use the dog as a natural model to ascertain metabolic and genetic risk factors for urolithiasis. Cystine and urate stones have previously been reported to have shared susceptibility genes between dogs and people. This thesis demonstrated that 2,8-dihydroxyadenine urolithiasis provides a third example of stone disease with a shared genetic basis between the species. It is likely that there are also overlapping genetic risk factors for CaOx urolithiasis. Prior to initiating genetic investigations into CaOx urolithiasis, three canine breeds were evaluated for metabolic disturbances associated with stone risk. Each of the breeds was shown to have idiopathic hypercalciuria, the trait underlying stone risk in people. Two breeds were subsequently selected for genome-wide association studies. Three unique susceptibility loci were identified. This supports a polygenic basis for CaOx stone risk in dogs. All three loci harbor plasma membrane transporter genes. One locus on CFA37 was selected for variant discovery using whole genome next-generation sequencing. No putative causal mutations were identified in coding sequence for the positional genes, but markers in and near the top candidate gene, SLC39A10, were associated with disease in a large cohort. SLC39A10 encodes a plasma membrane metal ion transporter. Several metals have been debated as potential triggers of stone formation. Genotype for the SLC39A10 risk haplotype was found to correlate with urinary strontium and calcium levels, as well as stone risk. This provides evidence that dogs with the SLC39A10 risk haplotype may have a functional mutation in the gene that directly or indirectly alters handling of strontium and/or calcium. Though no coding variants were found in SLC39A10, the mutation could reside within a regulatory region of the gene. Future sequencing and expression studies are planned to further evaluate SLC39A10 and genes within the other risk loci for a role in CaOx urolithiasis.
Molecular bases of equine polysaccharide storage myopathies
(2015-04) Teixeira, Raffaella
Polysaccharide Storage Myopathy (PSSM) is a form of glycogen storage disease in horses, characterized by abnormal polysaccharide inclusions in skeletal muscle. PSSM1 is caused by a dominant gain of function mutation in the GYS1 gene. PSSM1 horses can metabolize glycogen and have a normal flux of metabolites through glycolysis during maximal exercise, yet these horses demonstrate exercise intolerance, painful muscle cramping, and rhabdomyolysis during sub-maximal exercise. The link between excessive muscle glycogen, abnormal polysaccharide and rhabdomyolysis during sub-maximal exercise is less clear. To evaluate the changes in muscle that lead to this energy deficit, muscle gene expression profiles before and after a controlled exercise trial were evaluated in PSSM1 cases and controls by RNASeq. 201 genes were differentially expressed between cases and controls pre-trial, 301 genes were differently expressed between cases and controls pre-exercise (end of trial) and 803 genes were differentially expressed between cases and controls post-exercise (end of the trial). Gene set enrichment analysis revealed enrichment in pathways involving mitochondria biogenesis, oxidative phosphorylation, fatty acid metabolism, glycogen and glucose metabolism. DAVID was used to cluster the top differentially expressed genes based on their functional annotation. Clusters involved in inflammation were overrepresented. A second form of PSSM (PSSM2) has also been described. The mutation responsible for PSSM2 is unknown. Genome wide association data revealed significant markers associated with PSSM2 on equine chromosome 18 but no variants associated with PSSM2 were identified after extensive investigation of this region using a combination of target and whole genome sequencing in cases and controls. Imputation was then performed to increase the number of SNP markers in the initial GWA from 54,000 to close to 1.8 million markers, revealing new regions associated with PSSM2 on chromosomes 27 and 11. Haplotype analysis supported the association only on chromosome 11. The region of ECA11 encompasses several annotated genes. Next generation sequencing data from cases and controls revealed non-synonymous mutations in the phosphoribosylformylglycinamidine synthase gene in 2 out of 3 cases and none of the controls, identifying a new region in which to focus our efforts to define the genetic basis for PSSM2.
Pleiotropy and epistasis in trans-acting expression quantitative loci hotspots
(2023-11) Van Dyke, Krisna
Differences in non-coding regions of genomes explain the majority of heritable differences between individuals such as disease heritability. These non-coding differences are thought to largely act by altering gene expression, positioning regulatory variation as a key bridge between DNA variation and differences in traits. Expression quantitative trait loci (eQTLs) are regions of the genome containing one or more variants that alter the expression of a gene. In a cross between two strains of Saccharomyces cerevisiae, most heritable variation in gene expression acted in trans, with 90% of these trans-eQTLs overlapping only 102 “hotspot” loci. The large amount of heritable variation in gene expression that hotspots account for, and their discovery across the tree of life suggest they are a critical and ubiquitous feature of genome architecture. Classifying the structure of genetic variation underlying hotspots and learning what mechanisms allow hotspots to affect such large numbers of genes is critical to understanding how genetic variation gives rise to phenotypic variation. The following chapters describe a dissection of the variation underlying a hotspot and the uncovering of a new framework for how hotspots affect such numerous genes. Chapter II details how hotspots can co-opt the cellular mechanisms that cause adjacent genes to be coexpressed to extend their effect in cis. Chapter III dissects a hotspot with a complex epistatic basis to demonstrate how variants and groups of variants within a gene can interact to have wide-reaching impacts on the expression of many genes.
Ploidy, Genetics, And Metabolomics Of Achillea, A Venerable And Variable Medicinal Plant
(2022-09) Sammons, Katherine
Humans have turned to plants for medicine since ancient times. In recent centuries technology has allowed isolation of pure compounds which largely replaced herbal medicines in the USA around the beginning of the 20th century. However, even in today’s world of modern medicine, some herbal medicines can offer support for health in ways that pharmaceutical drugs cannot. With the advent of high resolution analytical instruments it is now possible to reconsider herbal medicines in all of their chemical complexity. Using the ancient medicinal plant Achillea millefolium as a case study, I use high resolution accurate mass-mass spectrometry to chemically profile 115 publicly available accessions of Achillea spp. cultivated in a common garden.In Chapter 1 I describe a brief history of medicine and technology to understand how we arrived at the current reductionist single-active-constituent mindset, and why it is important to broaden our mental model. In Chapter 2 I use flow cytometry and karyology to describe the ploidy of each of the 115 Achillea accessions. I also use DArTseq, a SNP analysis technique, to describe the population structure among these accessions. Finally, in Chapter 3 I use high performance liquid chromatography tandem to a high resolution mass spectrometer to chemically profile the 115 accessions. Altogether, this study helps to better understand the role that ploidy and genotype play in the chemotype of Achillea spp. The high resolution chemical profiling also helps to re-envision plants with a higher level of chemical complexity. Additionally, it serves as a potential model for improving approaches to quality control in the herbal industry.
Role of coat color genotypes in risk and severity of melanoma in gray Quarter Horses
(2013-06) Teixeira, Raffaella
Both graying and melanoma formation in horses have recently been linked to a duplication in the syntaxin-17 (STX17) gene. This duplication, as well as a mutation in the agouti signaling protein (ASIP) gene that increases melanocortin-1-receptor (MC1R) pathway signaling, affect melanoma risk and severity in gray horses. We hypothesized that melanoma susceptibility in gray Quarter Horses (QH) is lower than gray horses from other breeds, and that this might be due to decreased MC1R signaling resulting from a high incidence of the MC1R chestnut coat color allele in the QH population. Blood or hair root samples were collected from 335 gray QH with and without dermal melanomas, for DNA extraction and genotyping for STX17, ASIP and MC1R genes. Age, gender and external melanoma presence and grade were recorded. The effect of age and genotype on melanoma presence and severity was evaluated by candidate gene association study. The melanoma prevalence and grade in this QH cohort were lower than in other breeds. Age was significantly associated with melanoma prevalence and severity. No significant effect of MC1R genotype on melanoma prevalence or severity was identified. In contrast to prior reports, an effect of ASIP genotype on both melanoma prevalence and grade was not detected. Homozygosity of STX17 was low and precluded evaluation of the gray allele effect on melanoma presence and severity. Melanoma prevalence and severity appears to be lower in gray QH than in other breeds. This could be due to infrequent STX17 homozygosity, a mitigating effect of the MC1R mutation on ASIP potentiation of melanoma, other genes in the MC1R signaling pathway, or differences in breed genetic background.
Understanding the genetic architecture of secondary domestication traits in Field Pennycress (Thlaspi arvense L.)
(2022-12) Tandukar, Zenith
Thlaspi arvense L. (Field Pennycress) is a newly domesticated winter annual oilseed capable of improving ecosystems and intensifying agricultural productivity without new land displacement. Pennycress is a winter hardy cover crop that provides ecosystem services such as reduced soil erosion and nutrient loss in between fall corn harvest and spring soybean planting. However, pennycress is currently limited by its small seed size and unimproved oil production. This dissertation builds on the limited research on pennycress breeding and genetics and aims to establish and characterize a global diversity panel of wild pennycress accessions, two biparental recombinant inbred populations, and three independent EMS-derived mutants to contribute knowledge and resources to understand important seed and agronomic characteristic traits in pennycress. Chapter 1 presents a literature review focused on the status of pennycress breeding and genetics, as well as factors that may shed light to understanding the genetic and physiological control of seed size and oil content. Chapter 2 presents a genetic dissection of seed size, oil content, and protein content via genome-wide association studies in a diversity panel. Chapter 3 explores and characterizes the phenotypic and genotypic diversity in two recombinant inbred populations developed for field pennycress, whereas chapter 4 reports the characterization of three independent wax mutants in pennycress and the implications of waxes on total seed oil content in pennycress.

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