>Magister Scientiae - MSc / The human arylamine N-acetyltransferase 1 (NAT1) is critical in determining the duration of action and pharmacokinetics of amine-containing drugs such as para-aminosalicylic acid and para-aminobenzoyl glutamate used in clinical therapy of tuberculosis (TB), as well as influencing the balance between detoxification and metabolic activation of these drugs. SNPs in this enzyme are continuously being detected and indicate inter-ethnic and inter-individual variation in the enzyme function. The effect of nsSNPs on the structure and function of proteins are routinely analyzed using SIFT and POLYPHEN-2 prediction algorithms. The false-negative rate of these two algorithms results in as much as 25% of nsSNPs. This study aimed to explore the use of homology modeling including residue interactions, Gibbs free energy change and solvent accessibility as additional evidence for predicting nsSNP effects on enzyme function.This study evaluated the functional effects of 14 nsSNPs identified in a South African mixed ancestry population of which 3 nsSNPs were previously identified in Caucasians. The SNPs were evaluated using structural analysis that included homology modeling, residue interactions, relative solvent accessibility,Gibbs free energy change and sequence conservation in addition to the routinely used nsSNP function prediction algorithms, SIFT and POLYPHEN-2. The structural analysis implemented in this study showed a loss of hydrogen bonds for S259R thereby affecting protein function which contradicts predictions obtained from SIFT and POLYPHEN-2 algorithms. The variant N245I was shown to be neutral but contradicted the predictions from SIFT and POLYPHEN-2. Structural analysis predicted that variant R242M would affect protein stability and therefore NAT1 function in agreement with POLYPHEN-2 predictions but contradicting predictions from SIFT. No structural changes were expected for variant E264K in agreement with predictions obtained from POLYPHEN-2 but contradicting results from SIFT. The functions of the remaining 10 nsSNPs were consistent with those predicted by SIFT and POLYPHEN-2 namely that four variants R117T, E167Q, T193S and T240S do not affect the NAT1 function whereas R166T, F202V, Q210P, D229H, V231G and V235A could affect the enzyme function.This study provided the first evaluation of the functional effects of 11 newly characterized nsSNPs on the NAT1 tuberculosis drug-metabolizing enzyme. The six functionally important nsSNPs predicted by all three methods and the four SNPs with contradictory results will be tested experimentally by creating a SNP construct that will be cloned into an expression vector. These combined computational and experimental studies will advance our understanding of NAT1 structure-function relationships and allow us to interpret the NAT1 genetic polymorphisms in individuals who are slow or fast acetylators. The results, albeit a small dataset demonstrate that the routinely used algorithms are not without flaws and that improvements in functional prediction of nsSNPs can be obtained by close scrutiny of the molecular interactions of wild type and variant amino acids.

>Magister Scientiae - MSc / The primary objective of this Masters project was to investigate the maternal ancient substructure of the Griqua population in South Africa. Genetic ancestry was determined by investigating ancestry informative single nucleotide polymorphisms. These are located in the control region of the mitochondrial genome. The auxiliary aim was to test the validity of the UWC 10plex system in relation to a sample group of Griqua males. This short tandem repeat multiplex targets specific mutations confined to paternal lineages. The Khoi Khoi or Hottentots were the first inhabitants in the Cape. Indigenous Khoi Khoi female slaves had offspring with the European settlers in the 1800s which resulted in the Griqua population group. The incorporated European paternal ancestry is what set the Griqua apart from the native population groups at that time. Colonisation events from the mid-17th to 19th Century and the apartheid regime resulted in land dispossession of the native population and an extensively mixed gene pool in South Africa. One hundred and seventy six (N=176) male and female Griqua people were collectively sampled in Kokstad (2012), Vredendal (2012 and 2013) and at the Griqua National Conference in Ratelgat (2013). All 176 samples were analysed using mtDNA control region Sanger sequencing. The sample group (N=176) was separated based on birthplace (Origin sample group and post-colonial sample group). The origin sample group consists of individuals whose ancestors were not part of the Griqua Trek to Northern regions of South Africa and were less likely to be exposed to colonial influences. Mutations within the hypervariable segments of the mtDNA control region were used to infer haplogroups with geographic-specific population data. In this way one can plot the extent of ancient Khoisan (L0d) and Bantu influences (L1-L5) as well as the influence of East (M, A, B, E) and West (N, R, J, H) Eurasian haplogroups in the maternal ancestry of the Griqua population group. The origin sample group showed 91% African ancestry (76.8% L0d) while the post-colonial group had 78% African ancestry (60% L0d). The origin sample group had 2% East Eurasian and 7% West Eurasian ancestry, while the post-colonial group contained 20% Eurasian ancestry. There is greater admixture in the post-colonial group which can be attributed to the integration of surrounding populations during settlement periods in parts of the Northern Cape and KwaZulu-Natal. The UWC 10plex STR kit was tested to see if it could discriminate between male individuals of this admixed sample group (N=91 males). The markers for this multiplex were selected according to their ability to differentiate between individuals of African descent. It proved to be a viable Y chromosome short tandem repeat testing tool, displaying a statistically significant discrimination capacity value of 0.966 and only having 3 shared haplotypes in the sample group of 91 Griqua males. / National Research Foundation (NRF)

>Magister Scientiae - MSc / Genetic variability in genes encoding drug metabolizing enzymes, transporters and targets are known to be the main factors of inter-individual differences in therapeutic outcome. Genetic factors are estimated to be responsible for about 15-30% of inter-individual variation in drug disposition and response. Single-nucleotide polymorphisms (SNPs) are the most prevalent class of genetic variation that could explain the variability in drug efficacy and undesired side effects for patients. The aims of this study were to develop and evaluate the performance of robust and high throughput techniques for genotyping ten polymorphisms related to anticancer drugs and ten polymorphisms related to cholesterol lowering drugs. SNaPshot minisequencing and high resolution melt analysis (HRM) genotyping panels were developed, optimized, and their performances were evaluated and compared. SNaPshot minisequencing systems were developed and successfully optimized for the genotyping of ten SNPs associated with anticancer drug therapy, and ten SNPs associated with cholesterol lowering drugs. These systems were used to genotype the selected SNPs in 130 healthy Cape Admixed participants residing in Cape Town, South Africa. Population genetics data obtained for the studied SNPs were analysed using several statistical analysis software tools. Important population genetic parameters were calculated. Among others, allelic and genotypic frequencies were determined and compared with other populations in the world. High resolution melt analysis (HRM) genotyping panels were developed, optimized and their performance were evaluated and compared to the SNaPshot assays. HRM was explored as an alternative inexpensive and rapid methodology to genotype five SNPs related to anticancer therapy and five SNPs related to cholesterol lowering therapy (statins). Unlike the SNaPshot assays, rigorous optimization was required for the detection heterozygous genotypes via HRM. Both assays were validated using direct sequencing and compared to each other. The HRM system is a closed tube, cheap and (theoretically) rapid method for identifying genetic variations. HRM was however found to be more time consuming, needed further optimization, primer redesigning and more evaluation. The developed genotyping systems could be further validated using clinical samples from patients. This could help in optimizing drug therapy for cancer and cholesterol treatment.

Background: Hypertension, a leading risk factor for cardiovascular disease, exhibited in 17.7% of the Canadian population, and attributed to 13% of world mortality, is influenced by both the environment and genetics. Salt sensitivity is described at higher rates in the hypertensive population. The NEDD4-like (NEDD4L) protein is important in sodium reabsorption and has been implicated in essential hypertension and salt sensitivity. Objectives: Two variations (rs4149601/rs2288774) found in NEDD4L have been associated with salt sensitivity and hypertension; a third (rs576416) is in linkage disequilibrium with rs4149601. The purpose of this study is to assess the relationship between the NEDD4L rs4149601, rs2288774, and rs576416 single nucleotide polymorphisms with sodium and age on blood pressure (BP). Methods: Canadian hypertensive patients were recruited through the University of Ottawa Heart Institute, with genotyped data from Leuven, Belgium, and the DNA of subjects from Warsaw, Poland also included in the study. Eligible subjects were studied off anti-hypertensive medications. Daytime BP was measured using 24hr ambulatory BP monitoring in 662 Caucasian hypertensives (BP ≥130/85 mmHg). 24hr urine Na+ was collected. DNA from Canada and Poland was genotyped on the GeneTitan Affymetrix Axiom platform and through TaqMan MGB probe-based RT-PCR, while the Belgium samples were analyzed on Illumina 1M-duo arrays. Simple and multivariate linear regression modelling with SAS 9.4.0 was used for genotypic comparisons affecting BP, combined with age and corrected urine sodium. Results: The three hypertensive populations were significantly different (P<0.05) across all demographic and clinical measures, even when stratified by sex. The Polish and female hypertensives from Canada and Belgium were removed from the analysis for lacking the general populations’ trend of increasing BP with age. Multiple linear regressive modelling found a significant association (Pmodel=0.0034) of rs4149601 GA (P=0.0129) and GG (P=0.0082), with age and urine sodium, on SBP in the Belgium male hypertensives (n=273). No significant models analyzing the association of rs576416 and rs2288774 with BP in the Belgium population were found. In the Canadian hypertensive population (n=120) no association on the discrete analyses of the rs4149601, rs576416, and rs2288774 genotypes were found; however the combination of the GG rs4149601 and AA rs576416 (β=0.021, P=0.03) and the GG rs4149601 and CC rs2288774 (β=0.020, P=0.04) genotypes showed significant associations with BP in borderline significant models (P=0.055 and P=0.094 respectively), when analyzed with urine sodium levels and age. Conclusions: A significant influence of the rs4149601 G-allele, with urine sodium and age, was found to be associated with an increase in BP in the Belgium males. Multiple linear modelling describing borderline significant findings in the interaction of rs4149601 with rs576416, and rs4149601 with rs2288774 in Canadian male hypertensives suggests of the possible synergism between polymorphisms and development of salt sensitive hypertension. Future research could evaluate the role of NEDD4L on the sex differences in early-onset salt-sensitive hypertension.

Reactive oxygen species are formed internally through cellular metabolism and through external sources including radiation and pollutants. They play an important role in physiologic functions; however, when reactive oxygen species exceed our body’s antioxidant defense system, oxidative stress can occur. Oxidative stress has been implicated in aging and aging-related diseases including cancer and cardiovascular disease. Numerous oxidative stress genes produce antioxidative enzymes to mitigate the effects of reactive oxygen species. Single nucleotide polymorphisms within these genes may impact the functionality of antioxidant enzymes produced leaving the body more susceptible to damage from oxidative stress. The Iowa 65+ Rural Health Study was one of the four study populations in the Established Population for Epidemiologic Studies of the Elderly (EPESE) project initiated by the intramural Epidemiology, Demography and Biometry Program of the National Institute on Aging in 1980. The Iowa cohort was comprised of Iowa county and Washington county residents aged 65 and older at the time of the baseline interview in 1982. Participants completed three in-person interviews and five telephone interviews over eight years which collected data on habits, lifestyle and disease. During the in-person Year 06 interview participants were asked to donate a blood sample. The DNA extracted from the samples was used in each of the three aims of this project. The first aim evaluated single nucleotide polymorphisms in selected oxidative stress genes and their association with lifespan while controlling for aging-associated risk factors such as body mass index, comorbidity, alcohol consumption, smoking, and physical activity. Multivariable linear regression models were fit in the framework of the co-dominant genetic model. The oxidative stress genes selected for this project included the sirtuin family of genes (SIRT1-7), two of the forkhead box genes (FOXO1 and FOXO3), superoxide dismutase 2 and 3 (SOD2 and SOD3), glutathione peroxidase (GPX1), AKT, TP53, and CAMK4. A model was fitted with the risk factors before assessing the impact of each single nucleotide polymorphism. The q-value was used to control for the multiple hypothesis tests. Significant associations were detected between human lifespan and SNPs in genes SIRT3, SIRT5, SIRT6, FOXO3, and SOD3; gender modified the effect of SNPs in SIRT3, SIRT5, and AKT1. The second aim of this project evaluated single nucleotide polymorphisms in selected oxidative stress genes and their association with blood pressure measures while controlling for known risk factors including body mass index, alcohol consumption, smoking, and physical activity. Blood pressure was measured at the baseline and Year 06 interviews. Systolic pressure and diastolic pressure were used to calculate mean arterial pressure and pulse pressure at baseline and Year 06. Multivariable linear regression was used within the co-dominant genetic framework to determine if single nucleotide polymorphisms in SIRT1-7, FOXO1, FOXO3, SOD2-3, GPX1, AKT, TP53, and CAMK4 were associated with systolic, diastolic, mean arterial, or pulse pressure at baseline or Year 06. To examine longitudinal effects, the difference between each measure (i.e., Year 06 systolic – baseline systolic) was calculated for each individual and used to evaluate if any of the single polymorphisms was associated with change in blood pressure measures over time. Significant associations were detected between SIRT1 and SIRT3 and for males in SIRT1 and various blood pressure measures for females. Gender modified the effect of SIRT1, SIRT3, SIRT6, and FOXO1 variants. The third aim of this project evaluated if these genetic variants were associated with incident stroke while controlling for known risk factors including blood pressure, diabetes, body mass index, alcohol consumption, smoking, and physical activity. Multivariable logistic regression within the framework of the co-dominant genetic model was used. Individuals with the GPX1 genotype TT had 2.76 times the risk of an incident stroke compared to the CC genotype. This project identified several associations between single nucleotide polymorphisms within oxidative stress genes and lifespan, blood pressure measures, and incident stroke. Gender modified the association of several single nucleotide polymorphisms and lifespan as well as blood pressure measures. These results suggest genetic variation within oxidative stress genes may play a role in aging, blood pressure and incident stroke.

Plasmodium is a genus well known for causing malaria, a life-threatening infection for many people where malaria is endemic. The blood-borne disease is transmitted by the female Anopheles mosquito. Till date, eight parasite species have been reported to cause malaria in humans that include P. falciparum, P. vivax, P. malariae, P. ovale curtisi, P. ovale wallikeri, P. cynomolgi, P. knowlesi and more recently P. simium. Amongst them, the most genetically understood species is P. falciparum, causing most of the deaths in children from malaria. Understanding genome variation at the population level of all malaria species is of utmost importance, including clinical cases with very low parasitemia. To achieve this purpose, we need sufficient amounts of parasite DNA material from the pool of host DNA, which always is overrepresented in clinical infections. We utilized a strategy of selective whole genome amplification (SWGA) technology on P. malariae and P. ovale curtisi (two neglected human infecting malaria parasites that often cause mild yet clinically relevant infections with low parasitemia) to efficiently enrich their genomic DNA for high-quality whole genome sequencing. Previous studies on SWGA applied on P. falciparum and P. vivax showed that SWGA could efficiently enrich the amount of starting DNA material from inadequate amounts of parasites directly from clinical samples without separating the host DNA using specifically designed primer sets. We have successfully designed multiple sets of primers and tested the efficiency of five best primer sets using polymerase chain reaction to enrich the genomes of P. malariae and P. ovale curtisi. The efficiency of primers in enriching the genome was tested on two clinical samples for each of P. malariae and P. ovale curtisi. We were able to enrich the genome of P. malariae with an average of 19-fold (19X) enrichment across both samples. For P. ovale curtisi, we could achieve an enrichment of 3 folds only. Nevertheless, we still obtained a sufficient amount of gDNA to prepare Illumina sequencing libraries and call for SNPs and Indels in a biologically reproducible manner at genome-scale.

<p>Due to Ireland’s secluded geographical location, its genetic structure is a popular topic of study. The indigenous inhabitants of Ireland remained undisturbed for a long period time, allowing for a distinct genetic population to be created. This peace was disrupted by conflict with invading forces, such as the Nordic Vikings and Anglo-Norman forces. However, these historical events helped to shape both the culture of Ireland and the ancestry seen in the Irish population today. In Ireland, quite like many countries around the world, the male’s surname is passed from father to son, just as the Y-chromosome. The relationship between Irish surnames and their corresponding Y-haplogroups was examined to determine if common and rare Irish surnames can be genetically linked to the historical invasions listed above. The surnames chosen for this study were selected based on their prevalence in Ireland, rare or common, and their proposed historical origin, Irish, Norse or British. To discover any possible patterns in surnames and Y-chromosomal DNA, Y-haplogroups were generated from the DNA of 630 Irish male subjects using an assay specifically developed for the region. The assay contains twenty single-nucleotide polymorphisms (SNPs) that were selected to further resolve the R1b-L21 Y-haplogroup for Irish ancestry, the most prevalent haplogroup in Western Europe, and Ireland in particular. Additional Y-STR data was also generated to examine recent surname history within the collected individuals. Each surname was examined to determine whether one haplogroup occurred more frequently and with this method, distinct patterns in Irish surnames and geographical locations were discovered. In addition to resolving Y-surname history patterns, it is also believed that this assay may be beneficial in determining if an unknown DNA sample is of Western European origin and even in some cases, if a more specific Irish origin can be predicted.</p>

>Magister Scientiae - MSc / Anxiety, resilience and childhood trauma can be categorized as functional behavioural categories, with a wealth of research behind each. The research approach adopted for each, in most cases, is either from a genetic or neuropsychological standpoint, with few studies combining both to better understand all three functional behavioural categories as a multidimensional construct A number of candidate genes have been identified as markers for anxiety, resilience and childhood trauma, of which Catechol-methyl-transferase (COMT) and several respective single nucleotide polymorphisms (SNPs) are included. Although COMT SNPs have been linked to at least one of the functional categories, with a handful of haplotypes identified, to our knowledge no study has investigated the combination of SNPs selected for this study (rs6269, rs4818, rs4680, rs4633, rs737865, rs2075507) as a possible haplotype, specifically in a South African population. The use of SNaPshot for the genotyping of genes is an efficient and reliable means of identifying genotype frequencies and haplotypes in large sample groups, yet when selecting more than two SNPs of interest, the development of a multiplex assay is ideal. The first aim of the study was to design and optimize a multiplex assay to genotype several COMT SNPs. The primer design, multiplex optimization and SNaPshot conditions used showed good working parameters that can be utilized and further improved by optimization. Self-report measures are widely used to measure psychiatric disorders, such as anxiety, and has also been used for the measurement of resilience and childhood trauma. With each functional behavioural category well investigated in its respective domain, there is a need to investigate all three as a collective in a South African population due to the high rate of anxiety and childhood trauma exposure in communities. The second aim of the study was to investigate the prevalence of anxiety, resilience and childhood as functional behavioural categories in the full South African sample group; and the role of sex, through established self-report measures and respective normative data. Additionally, this carried over into investigating the correlation between anxiety, resilience and childhood trauma as a multidimensional construct in both the full South African sample and between sexes. There is a clear relationship which exists between all three functional behavioural categories, as they show a correlation in various dimensions independent of one another. Higher anxiety levels amongst females were reported, with no difference between sexes for resiliency. The empirical data collected from both COMT SNP and self-report measures for male and female where explored and reviewed against current literature for better understanding and insight into the association of COMT SNPs with anxiety, resilience and childhood trauma in a South African population. The results of this study to understand the complexity and association of all three functional behavioural categories as a multidimensional construct will be invaluable and may assist in the identification of possible risk factors which are essential for the promotion of better mental health in society.

The Western Massasauga (Sistrurus tergeminus) is a small, North American rattlesnake found west of the Mississippi River. Sistrurustergeminushas previously been divided into two putative subspecies, Desert (S. t. edwardsii) and Prairie Massasaugas (S. t. tergeminus) based upon qualitative variation in morphology, coloration, and habitat. The Desert Massasauga subspecies has been formally petitioned for federal listing under the U.S. Endangered Species Act. Ouroverarching goal was to evaluate genetic structure and genomic differentiation between specimens of the two putative subspecies in an effort to inform ongoing conservation assessments. To that end, we generated whole genome sequence data for both putativetaxa and then developed nearly 200 genetic markers from different fractions of the genome (~50 intergenic and ~50 genic markers from each of the two subspecies) to test for population structure across much of the Western Massasauga range. Mean genomic divergence between subspecies was only 0.0041 ± 0.0080 (Kimura’s 2-parameter distance) for nuclear sequences and 0.0175 ± 0.0031 for mitochondrial sequences, both exceedingly low values which approach the minimum of zero. Admixture analyses and F-statistics both indicated that regardless of how the markers were partitioned, genetic structure was oriented far more along a geographic axis (isolation-by-distance) than a taxonomic axis (i.e., between putative subspecies). Overall, our analyses provide little support that formal protection of the purported Desert Massasauga is warranted based on the homogeneity of the collective Western Massasauga gene pool.

Alzheimer’s disease (AD) is a multifactorial neurological condition associated with genetic profiles that are still not completely understood. We performed a family-based low-density genome-wide association analysis of age at onset (AAO) in AD (244 patients and their relatives) using Illumina 6 K single-nucleotide polymorphisms (SNPs) panel and the FBAT-logrank statistic. We observed 10 SNPs associated with AAO in AD with p < 2 × 10−3. The most significant hit within a known gene, the neuronal protein astrotactin 2 (ASTN2), was SNP rs1334071 (p = 8.74 × 10−4). ASTN2 has been implicated in several neuropsychiatric disorders, including cognitive disorders, autism and schizophrenia. We then conducted a replication study focusing on ASTN2 gene in a Canadian sample of 791 AD patients and 782 controls using the logrank test. Five ASTN2 SNPs (highest association is rs16933774 with p = 0.0053) showed associations with AAO in this Canadian sample (p < 0.05). Furthermore, Kaplan–Meier survival analysis of SNP rs16933774 showed that the AAO of AD in individuals heterozygous for AG genotype of rs16933774 (median of AAO = 68.5 years) was approximately 4.5 years earlier than those individuals having the AA genotype (median of AAO = 73 years). In conclusion, a significant association of ASTN2 genetic variants with AAO of AD in two independent samples demonstrates a role for ASTN2 in the pathogenesis of AD. Future functional studies of this gene may help to characterize the genetic architecture of the AAO of AD. Genetic factors in AAO may be a critical factor for early AD intervention and prevention efforts.