Single nucleotide polymorphisms (SNPs) are single base variations among groups of individuals. In order to study their properties in fine gene mapping, I considered their occurrence as transitions and transversions. The aim of the study was to classify each polymorphism depending upon whether it was a transition or transversion and to calculate the proportions of transitions and transversions in the SNP data from the public databases. This ratio was found to be 2.35 for data from the Whitehead Institute for Genome Research database, 2.003 from the Genome Database, and 2.086 from the SNP Consortium database. These results indicate that the ratio of the numbers of transitions to transversions was very different than the expected ratio of 0.5. To study the effect of different transition to transversion ratios in fine gene mapping, a simulation study was performed to generate nucleotide sequence data. The study investigated the effect of different transition to transversion ratios on linkage disequilibrium parameter (LD), which is frequently used in association analysis to identify functional mutations. My results showed no considerable effect of different transition to transversion ratios on LD. I also studied the distribution of allele frequencies of biallelic SNPs from the Genome Database. My results showed that the most common SNPs are normally distributed with mean allele frequency of 0.7520 and standard deviation of 0.1272. These results can be useful in future studies for simulating SNP behavior. I also studied the simulated data provided by the Genetic Analysis Workshop 12 to identify functional SNPs in candidate genes by using the genotype-specific linkage disequilibrium method.

Background: Essential hypertension is regarded as a complex disease, the phenotype of which results from interactions between numerous genes and environmental factors. Genome-wide association studies of blood pressure (BP) and hypertension have been developed to explore the potential genes involved in blood pressure and identified a number of trait-associated variants. Among those variants, single nucleotide polymorphisms (SNPs) rs1173771 (G/A) and rs1421811 (G/C) are located at the natriuretic peptide receptor C (NPR3) gene locus. Their major alleles are related with blood pressure elevation. Studies have implicated NPR-C in mediating some of the cardio-protective actions of natriuretic peptides and its direct involvement in the pathogenesis of hypertension. However, the precise role of these association between genetic variants at NPR3 and blood pressure control has not been elucidated. Objective: To functionally characterise the effect of BP-associated SNPs at the NPR3 gene locus in the context of BP regulatory pathways. Methods: Primary human umbilical artery smooth muscle (HUASMCs) and vein endothelial (HUVECs) cells were genotyped for BP-associated NPR3 variants. Endogenous mRNA and protein expression levels were assessed by qRT-PCR, allelic expression imbalance assay and western blotting. Open chromatin regions were assayed using formaldehyde-assisted isolation of regulatory elements (FAIRE). Interaction between variants flanking region with nuclear protein was detected by electrophoretic mobility shift assay (EMSA). Cell proliferation and migration were 4 determined by cell counting and scratch assays. Angiotensin II (Ang II)-induced calcium flux was evaluated using the intracellular fluorescent probe. Results: The BP-elevating allele of the NPR3 variants in rs1173771 linkage disequilibrium (LD) block was associated with lower endogenous mRNA and protein levels in HUASMCs. This is consistent with the finding that BP-elevating allele is less located within open chromatin. The decreased NPR3 expression in HUASMCs carrying the BP-elevating allele is associated with increased cell proliferation and intracellular calcium flux in response to Ang II stimulation. No differences in migration rates were detected. No genotype-dependent characteristics were observed in HUVECs NPR3 expression and cell proliferation. Moreover, RT-PCR showed a linkage between of the BP-elevating allele of the NPR3 variants in rs1421811 LD block and lower endogenous mRNA in HUASMCs. Intracellular calcium flux detection also revealed a trend of higher response to Ang II stimulation in BP-elevating allele homozygous HUASMCs. However, No genetic differences were detected in proliferation and migration rates of HUASMCs, and HUVECs NPR3 expression and cell proliferation studies did not present any significant genotype-dependent association. Conclusions: This study has identified a potential mechanism for BP-associated SNPs at NPR3 locus to influence BP predominantly via an effect on vascular smooth muscle cell behaviours.

Type 2 diabetes and metabolic syndrome are the metabolic disorders which constitute a major public health problem in both developed and developing countries. Various studies have suggested the genetic susceptibility to the disorders. The main aim of the thesis was to investigate the putative association of single nucleotide polymorphisms with Type 2 diabetes (T2D), metabolic syndrome (MetS) and the major components of metabolic syndrome. This study used meta‐analysis, polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP) and Sanger sequencing methods to analyse the results. The single nucleotide polymorphism rs57829442 of peroxisome proliferator‐activated receptor‐γ coactivator‐1 (PPARGC1A) gene and its relation to risk of type 2 diabetes has been studied in the United Kingdom population. A meta‐analysis of genetic variant rs8192678 (Gly482Ser) of peroxisome proliferator‐activated receptor‐γ coactivator‐1 (PPARGC1A) gene and its association with the components of metabolic syndrome has been studied. An association of the genetic variants rs8192678 (Gly482Ser) of the PPARGC1A gene, rs7903146 of Transcription Factor 7 Like 2 (TCF7L2) gene, rs9939609 of Fat mass and obesity‐associated (FTO) gene and rs1801282 (Pro12Ala) of peroxisome proliferator‐activated receptor gamma (PPARG) gene with the metabolic syndrome and its components has been studied in the Nepalese population. The results showed that variant rs57829442 of PPARGC1A is not associated with T2D in the United Kingdom population. Further investigation with increased sample size is warranted. In the meta‐analysis, the variant rs8192678 (Gly482Ser) of PPARGC1A gene was found to be significantly associated with body mass index (BMI) in Asian populations under dominant genetic model, total cholesterol (TC) in non‐Asian population under recessive genetic model and with fasting plasma glucose (FPG) under a recessive model in overall and non‐Asian populations. No significant association of the variants rs8192678 (Gly482Ser), rs7903146, rs9939609 and rs1801282 (Pro12 Ala) was found associated with MetS under dominant, recessive, co‐dominant and additive models in the Nepalese population. However, the genotypes (AG and AA) of rs8192678 (Gly482Ser) had a statistically significant protective effect on systolic blood pressure. The genotypes with the risk allele of rs9930609 of FTO gene was significantly associated with weight, waist circumference and diastolic blood pressure under dominant genetic model and with BMI under both dominant and recessive genetic models in the Nepalese population. To the best of our knowledge, this is the first study to report the findings in the Nepalese population.

Familial hypercholesterolemias (FH) are inherited mutations that cause elevated total cholesterol and low-density lipoprotein cholesterol levels (LDL-C) which lead to premature coronary heart diseases. Pharmacogenetics is the study of inherited genetic differences in drug metabolic pathways which can affect the patient’s response to the drug. Single Nucleotide Morphism (SNP) mutations in the LDLR, apoB, LDRAP1, and PCSK9 genes are linked to familial hypercholesterolemia. The mutations in the LDLR gene are the most common while mutations in the apoB and PCSK9 genes are the least common in hypercholesterolemia patients. My research will study how pharmacogenetics can be used to diagnose and prescribe patients with FH who have apoB and PCSK9 double gene mutations. I will genotype and sequence the PCR amplified gene segments of the patients with FH to identify any of the 6 apoB SNPs and any of the 3 PCSK9 SNPs that are known. Then, I will provide 4 different treatments: placebo, antisense therapy (mipomersen), PCSK9 inhibitor (alirocumab), and a combination of mipomersen + alirocumab, and I will measure the LDL-C levels before and after a 12-week trial. I hypothesize that individuals with both apoB and PCSK9 gene mutations with the known SNPs that cause loss of function will be more responsive when given both treatments by observing a significant decrease in LDL-C levels.

Jun and Fos are components of the AP1 family of transcription factors that bind to the promoters of a diverse multitude of genes involved in critical cellular responses such as cell growth and proliferation, cell cycle regulation, embryonic development and cancer. The specific protein-DNA interactions are driven by the binding of basic zipper (bZIP) domains of Jun and Fos to TPA response element (TRE) and cAMP response element (CRE) within the promoters of target genes. Here, using a diverse array of biophysical techniques, including in particular isothermal titration calorimetry in conjunction with molecular modeling and semi-empirical analysis, I characterize AP1-DNA interactions in thermodynamic and structural terms. My data show that the binding of bZIP domains of Jun-Fos heterodimer to TRE and CRE are under enthalpic control accompanied by entropic penalty at physiological temperatures. This is in agreement with the notion that protein-DNA interactions are largely driven by electrostatic interactions and intermolecular hydrogen bonding. A larger than expected heat capacity change suggests that the basic regions within the bZIP domains are unstructured in the absence of DNA and interact in a coupled folding and binding manner. Further analysis demonstrates that Jun-Fos heterodimer can tolerate single nucleotide variants of the TRE consensus sequence and binds in the biologically relevant micromolar to submicromolar range. Of particular interest is the observation that the Jun-Fos heterodimer binds to specific variants in a preferred orientation. 3D atomic models reveal that such preference in orientation results from asymmetric binding and may in part be attributable to chemically distinct but structurally equivalent residues within the basic regions of Jun and Fos. I further demonstrate that binding of the biologically relevant Jun-Jun homodimer to TRE and CRE occurs with favorable enthalpic contributions accompanied by entropic penalty at physiological temperatures in a manner akin to the binding of Jun-Fos heterodimer. However, anomalously large negative heat capacity changes provoke a model whereby Jun loads onto DNA as unfolded monomers coupled with subsequent folding and homodimerization upon association. The data also reveal that the heterodimerization of leucine zippers is modulated by the basic regions and these regions may undergo at least partial folding upon heterodimerization. Large negative heat capacity changes accompanying the heterodimerization of leucine zippers are consistent with the view that leucine zippers do not retain a-helical conformation in isolation and the formation of the native coiled coil a-helical dimer is attained through a coupled folding-dimerization mechanism. Taken together, this dissertation marks the first comprehensive thermodynamic analysis of an otherwise well-studied and vitally important transcription factor. My studies shed new light on the forces driving the AP1-DNA interaction in thermodynamic and structural terms. The implications of these novel findings on the development of novel therapies for the treatment of disease with greater efficacy coupled with low toxicity cannot be overemphasized.

Rhesus macaques are a widely used animal model of human diseases and related immune responses. Fc receptors (FcRs) mediate the interaction between antibody molecules and innate killing mechanisms, consequently eliminating the pathogen. In rhesus macaques, FcRs are highly polymorphic. To evaluate the potential influence of FcgR polymorphisms on the interaction with antibody molecules, we performed in silico analysis using SIFT, Provean, nsSNPAnalyzer, I-Mutant, MuSTAB and iPTREE-STAB web servers. V20G in FcγRI, I137K in FcγRII and I233V in FcγRIII were further analyzed structurally using FOLD-X, AMMP and Chimera to calculate changes in folding and interaction energy and for structure visualization. Results from our analysis suggest that the selected variations destabilize protein structure. Additionally, Q32R increases the binding affinity of FcγRI, whereas A131T decreases the binding affinity of FcγRII towards IgG1. Together, our results indicate that these substitutions might influence effector and regulatory mechanisms resulting from antibody/FcR interactions.

DNA-metal ion interactions are critical for stabilizing conformations of double stranded (ds) DNA and through specific binding sites will influence the interaction of DNA with other molecules. It has been shown that different metal ions bind to different sites within nucleic acids. Work in this thesis exploits the interactions of Zn2+ with nucleic acids that are linked to surfaces. Zn2+ can interact with the phosphodiester backbone and engage in interactions with the purine nucleobases. Electrochemical studies of ds-DNA films have demonstrated that in the presence of Zn2+ films containing a single nucleotide mismatch give rise to a specific electrochemical signature. Electrochemical impedance spectroscopy (EIS) allows the discrimination of mismatched DNA films from those that are fully matched by monitoring differences in the resistance of charge transfer. Scanning electrochemical microscopy (SECM) allows multiplexing of the data acquisition and monitoring of the current response I, which is attenuated as a function of mismatch. In this thesis, various potential factors were explored in detail that may impact the discrimination of nucleotide mismatches in ds-DNA films by EIS and SECM. These factors include the position of the mismatch, its type, the number of mismatches, the length of the DNA duplex, and the length of target sequences. In particular, when the two strands are of unequal length, the resulting nucleotide overhang may mask the mismatch signature. Such overhangs are expected in real biosensor applications, in which the DNA is isolated from cellular targets. Results presented here clearly demonstrate that mismatches are readily distinguished from fully matched strands even in overhang systems, suggesting that this approach has promise for realistic sensor applications.

Abstract Primary lactose intolerance has been associated with a single nucleotide polymorphism located upstream of the lactase gene. The most common diagnostic tests for lactose intolerance are time-consuming and the patient is not allowed to eat and drink for 12 hours before the test is carried out. A method that can establish the genotype would be an easier way of diagnosing lactose intolerance compared to fenotypic lactose intolerance tests. Optimization and validation of a previously published method was performed with real-time polymerase chain reaction. We used whole blood from de-identified blood donors. During the optimization and validation we used a positive control, genotype C/T from Laboratoriemedicin Västernorrland, Sundsvall. The whole-blood was extracted using the MagNa Pure LC instrument. The reagent used was KAPA PROBE FAST qPCR Master Mix. The optimized program for real-time PCR was established to be 95°C 3min [95°C x 3sec, 55°C x 20sec, detection, 72°C x 15sec] x 50 cycles. Optimal probe concentration was found to be 0.2µM and primer concentration will be 0.5µM. This genotyping method is a good first-stage screening test for lactoseintolerance. Before it can be used as a routine method further validation will be necessary in order to ensure that the evaluation of the results can be done in an easy and secure way.

No description available.

Genomic data facilitate opportunities to track complex population histories of divergence and gene flow. We used 47,506 single-nucleotide polymorphisms (SNPs) to investigate cattle population history. Cattle are descendants of two independently domesticated lineages, taurine and indicine, that diverged 200,000 or more years ago. We found that New World cattle breeds, as well as many related breeds of cattle in southern Europe, exhibit ancestry from both the taurine and indicine lineages. Although European cattle are largely descended from the taurine lineage, gene flow from African cattle (partially of indicine origin) contributed substantial genomic components to both southern European cattle breeds and their New World descendants. We extended these analyses to compare timing of admixture in several breeds of taurine-indicine hybrid origin. We developed a metric, scaled block size (SBS), that uses the unrecombined block size of introgressed regions of chromosomes to differentiate between recent and ancient admixture. By comparing test individuals to standards with known recent hybrid ancestry, we were able to differentiate individuals of recent hybrid origin from other admixed individuals using the SBS metric. We genotyped SNP loci using the bovine 50K SNP panel. The selection of sites to include in SNP analyses can influence inferences from the data, especially when particular populations are used to select the array of polymorphic sites. To test the impact of this bias on the inference of population genetic parameters, we used empirical and simulated data representing the three major continental groups of cattle: European, African, and Indian. We compared the inference of population histories for simulated data sets across different ascertainment conditions using F[subscript ST] and principal components analysis (PCA). Ascertainment bias that results in an over-representation of within-group polymorphism decreases estimates of F[subscript ST] between groups. Geographically biased selection of polymorphic SNPs changes the weighting of principal component axes and can bias inferences about proportions of admixture and population histories using PCA. By combining empirical and simulated data, we were able to both test methods for inferring population histories from genomic SNP data and apply these methods to practical problems. / text