>Magister Scientiae - MSc / Autism or autism spectrum disorder (ASD) is a very diverse neurological disorder that manifests specifically in children and infants between the ages of two to three years of age. An individual suffering is deemed as autistic and individuals suffering would be classed under the banner of ASD. It is observed that sufferers have impairment in their social and interactive skills. It has both genetic and environmental factors that contribute to its diversity and although the primary cause of autism is still unclear, scientist are investigating both factors. In this study we aimed to investigate the molecular genetics of autism in the South African (SA) population. This was done in two parts, a genetic association study and afunctional genomics (proteomic study). An association study of the 2 single nucleotide polymorphisms (SNPs) of the Cadherin 10 type II gene (CDH10) (rs4307059 and rs4327572) was investigated in the SA healthy and autistic population. The proteomic approach was used to determine the differential expression of genes of the healthy population and compared to the autistic population of African descent. In both parts of the project, objectives were achieved. The SNPs were successfully genotyped however no association was determined for autism in the SA population. The urine protein profiles with 1 dimensional (1D) and 2dimensional (2D) Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis (SDSPAGE)generated in this study has revealed the following proteins, Uromodulin, Vitelline membrane outer layer protein homologue, kinninogen-1, Alpha-1-Antitrypsin, Ig Kappa chain region C, and CD59 glycoprotein that require further investigation. The results indicated that six of the identified proteins were expressed in both groups but were found to be either quantitatively or statistically significant. However, a statistically significant difference was observed in the expression of one protein (Uromodulin) which was observed to be expressed in the healthy group but absent in the experimental group. However further investigation is required validation of these findings.

Single nucleotide polymorphisms (SNPs) have been increasingly popular for a wide range of genetic studies. A high-throughput genotyping technologies usually involves a statistical genotype calling algorithm. Most calling algorithms in the literature, using methods such as k-means and mixturemodels, rely on elliptical structures of the genotyping data; they may fail when the minor allele homozygous cluster is small or absent, or when the data have extreme tails or linear patterns. We propose an automatic genotype calling algorithm by further developing a linear grouping algorithm (Van Aelst et al., 2006). The proposed algorithm clusters unnormalized data points around lines as against around centroids. In addition, we associate a quality value, silhouette width, with each DNA sample and a whole plate as well. This algorithm shows promise for genotyping data generated from TaqMan technology (Applied Biosystems). A key feature of the proposed algorithm is that it applies to unnormalized fluorescent signals when the TaqMan SNP assay is used. The algorithm could also be potentially adapted to other fluorescence-based SNP genotyping technologies such as Invader Assay. Motivated by the SNP genotyping problem, we propose a partial likelihood approach to linear clustering which explores potential linear clusters in a data set. Instead of fully modelling the data, we assume only the signed orthogonal distance from each data point to a hyperplane is normally distributed. Its relationships with several existing clustering methods are discussed. Some existing methods to determine the number of components in a data set are adapted to this linear clustering setting. Several simulated and real data sets are analyzed for comparison and illustration purpose. We also investigate some asymptotic properties of the partial likelihood approach. A Bayesian version of this methodology is helpful if some clusters are sparse but there is strong prior information about their approximate locations or properties. We propose a Bayesian hierarchical approach which is particularly appropriate for identifying sparse linear clusters. We show that the sparse cluster in SNP genotyping datasets can be successfully identified after a careful specification of the prior distributions. / Science, Faculty of / Statistics, Department of / Graduate

Indiana University-Purdue University Indianapolis (IUPUI) / 2022-08-31

No description available.

The human pathogenic Cryptococcus neoformans species complex are agents of a common AIDS-defining disease, which causes about 181,000 deaths each year. There are several specific features distinguishing this species from other fungi, including the presence of a polysaccharide capsule and melanin pigment production, both of which contribute to its virulence. A large number of studies about this pathogen used two model strains JEC20 and JEC21. In these studies, these two strains are assumed to be “isogenic”, differ only at the mating type region. Consequently, their phenotypic differences, including virulence, have been attributed to this region. Here, we applied second-generation sequencing and bioinformatics tools to identify sequence polymorphisms between the two genomes. Beside the Mating Type locus, two other regions were found to contain high frequencies of SNPs. To further understand the effects of these loci on the phenotypic differences, four phenotyping assays (mating ability, melanin pigment production, capsule formation, and high temperature growth ability) were conducted on the recombinant progeny obtained from the cross between JEC20 and JEC21. In addition, genomic sequences of these progeny were obtained to identify the complete distributions of other SNPs among the strains. Finally, we identified several novel SNPs contributing to virulence-related traits in this species, which suggest that caution should be placed in attributing phenotypic differences to specific genomic regions in “isogenic” strains derived from classical breeding experiments. / Thesis / Master of Science (MSc) / Cryptococcosis is a globally distributed infection that is prevalent among immune-compromised individuals, such as HIV/AIDS patients. This disease can be attributed to a group of opportunistic fungal pathogens – Cryptococcus neoformans species complex. During the past century, significant resources have been put in an effort to understand its ecology, evolution, life cycle, pathogenesis and virulence factors, and molecular and cellular processes. Most of the laboratory-based studies have relied on two model strains assumed to differ only at the mating type locus. My thesis investigated this assumption and found there are several additional significant genetic differences between these two strains and that such differences contribute to the observed phenotypic differences between them. My results highlight the complexity of genotype-phenotype relationships and the continued evolution of strains even in lab environments in C. neoformans.

Dilated cardiomyopathy (DCM), a heart disease, affects many vertebrates including humans and poultry. The disease can be either idiopathic (IDCM) or toxin-induced. Idiopathic DCM often occurs without a consensus cause. Though genetic and other studies of IDCM are extensive, the specific etiology of toxin-induced is still unknown. Here, our objective was to compare the level of mRNA expression of two candidate genes including troponin T (cTnT) and phospholamban (PLN) using quantitative reverse transcription polymerase chain reaction (RT-PCR) in toxin-induced DCM affected and unaffected turkeys. Cardiac TnT and PLN were chosen because their spontaneous expression has been reported to be associated with IDCM. We also scanned these genes for single nucleotide polymorphisms (SNPs) that could be useful in evaluating their functions in the incidence and severity of toxin-induced DCM in turkeys. There were no significant differences between affected and unaffected birds in the expression of both cTnT and PLN. A total of 12 SNPs were detected in cTnT and PLN DNA sequences. One of the seven haplotypes detected in cTnT was the most frequent. Linkage analysis showed that cTnT gene was unlinked on the current turkey genetic map. Resources developed here, including SNPs, haplotypes, cDNA sequences, and the PCR-RFLP genotype procedure will be used for future investigations involving cTnT and PLN and toxin-induced DCM. / Master of Science

Breast cancer affects 1 in 8 women and can be deadly; yet when detected early enough it is often treatable. Thus, early detection of breast cancer is imperative to save lives. The success of early detection depends, in part, on being able to stratify risk. A new approach to determining risk involves identifying genetic variants that alter an individual’s risk for developing breast cancer. This thesis identified key functional candidates involved in breast cancer development, some of which have been verified by other studies. For a few of the functional candidates, further research needs to be done in order to determine the biological significance they play in the development of breast cancer. The functional candidates were identified by comparing SNPs in Linkage Disequilibrium with high risk SNPS—determined by GWAS—using histone modification markers to identify functional genomic elements in breast cell lines. The results yielded three top tier candidates and multiple second tier candidates. Further research should be done in order to assess the risk involved with these variants and the underlying biological mechanism. As genetic testing becomes more accessible to the public, the identification and understanding of these high risk variants will be an essential tool in preventing and treating breast cancer.

Thesis (MMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The aetiology of multiple sclerosis (MS) remains largely unknown due to the multifactorial nature of disease susceptibility determined by both environmental and genetic factors. Progress has been made in identifying the genetic component of MS , as well as the possible interactions with the environment. In this study single nucleotide polymorphisms (SNPs) in the FTO (rs9939609, Intron 1 T>A), MTR (rs1805087, 2756 A>G), MTRR (rs1801394, 66 A>G), MTHFR (rs1801133, 677 C>T and rs1801131, 1298 A>C) and COMT (rs4680, 472 G>A) genes involved in the methylation metabolic pathway were studied in the context of MS. The overall objective of this study was to elucidate the mechanism underlying raised homocysteine levels in MS patients. The specific aims were 1) to analytically validate high throughput real-time polymerase chain reaction (RT-PCR) genotyping assays for the 6 selected SNPs against direct sequencing as the gold standard for 2) possible integration into a pathology-supported genetic testing strategy aimed at improved clinical management of MS. The study population included a total of 114 unrelated Caucasian MS patients (98 females and 16 males) and 195 unrelated Caucasian control individuals without a diagnosis of neurological disease (128 females and 67 males). A novel finding of this study was that the risk-associated FTO rs9939609 A-allele was associated with raised homocysteine levels (p=0.003) in patients diagnosed with MS, but not in controls. Furthermore, homocysteine levels correlated significantly with bo dy mass index (BMI) (p=0.046) and total cholesterol levels (p=0.048). Both homocysteine (p=0.011) and BMI (p=0.017) were significantly reduced with increasing intake of folate in the diet, while high saturated/trans fat intake correlated significantly with increased BMI (p<0.001). High physical activity correlated with reduced BMI (p<0.006) in the study population, adjusted for age, gender and disease status. Daily intake of at least five fruit and vegetable portions and the COMT rs4680 (472 G>A) AA genotype had a favourable lowering effect on MS disability as assessed by the expanded disability status scale (EDSS) (p=0.035), while smoking increased MS disability significantly (p<0.001). All SNPs studied were found to be in Hardy-Weinberg equilibrium (HWE), with no significant differences detected between patients and control individuals in genotype distribution or allele frequencies. This study has shown for the first time that the underlying disease process of MS moderates the effect of the FTO rs9939609 polymorphism on homocysteine levels , which is consistent with the role of FTO in demethylation and epigenetic changes. Identification of FTO rs9939609 reinforces the importance of adequate folate intake in the diet that can be assessed accurately with use of the Medical History and Lifestyle Questionnaire applied in this study. Finally, the finding that raised homocysteine levels and BMI are significantly influenced by lifestyle factors such as diet and physical activity in our study cohort , offers a solution to counteract the detrimental effects of genetic risk factors contributing to the development of these established vascular risk factors for MS. Combining this information with FTO rs9939609 and COMT rs4680 genotyping may in future translate into a comprehensive pathology supported genetic testing strategy aimed at improved risk management and quality of life in MS patients. / AFRIKAANSE OPSOMMING: Die etiologie van meervoudige sklerose (MS) is grootliks onbekend as gevolg van die multifaktoriale aard van siekte vatbaarheid wat bepaal word deur beide genetiese en omgewingsfaktore. Vordering is reeds gemaak in die identifisering van die genetiese component van MS, asook moontlike interaksie met die omgewing. In hierdie studie is enkel nukleotied polimorfismes (SNPs) in die FTO (rs9939609, Intron 1 T > A), MTR (rs1805087, 2756 A> G), MTRR (rs1801394, 66 A> G), MTHFR (rs1801133, 677 C > T en rs1801131, 1298 A> C) en COMT (rs4680, 472 G > A) gene, wat betrokke is in die metilering metaboliese padweg, in die konteks van MS bestudeer. Die oorhoofse doel van hierdie studie was om die onderliggende meganisme betrokke by verhoogde homosisteïen vlakke in MS pasiënte uit te lig. Die spesifieke doelwitte was 1) om die analitiese geldigheid van die hoë deurvoer riëeltyd polymerase kettingreaksie (RT-PCR) genotipering metode soos toegepas vir die 6 geselekteerde SNPs te bevestig teen direkte DNA volgorde bepaling as die goue standaard, vir 2) moontlike integrasie in 'n patologie-gesteunde genetiese toetsing (PSGT) stategie wat gemik is op verbeterde kliniese hantering van MS. Die studiepopulasie bestaan uit 'n totaal van 114 nie-verwante Kaukasiese MS pasiënte (98 vroue en 16 mans) en 195 nie-verwante Kaukasiese kontroles sonder ‘n diagnose van neurologiese siektes (128 vroue en 67 mans). 'n Nuwe bevinding van hierdie studie was dat die risiko-verwante FTO rs9939609 A- alleel geassosieer was met verhoogde homosisteïen vlakke (p = 0,003) in pasiënte gediagnoseer met MS, maar nie in kontroles nie. Homosisteïen vlakke was verder beduidend geassosieer met liggaamsmassa-indeks (BMI) (p=0,046) en totale cholesterol vlakke (p=0.048). Beide homosisteïen (p=0,011) en BMI (p=0,017) het aansienlik verminder met 'n hoër inname van folaat in die dieet, terwyl 'n hoë versadigde/trans vet en koolhidrate inname beduidend gekorreleer het met 'n verhoogde BMI (p <0.001). Hoë fisiese aktiwiteit was gekorreleer met 'n verminderde BMI (p< 0.006) in die gekombineerde groep, aangepas vir die ouderdom, geslag en MS diagnose. Daaglikse inname van ten minste vyf vrugte en groente porsies en die COMT rs4680 (472 G>A) AA genotipe het 'n gunstige uitwerking op vermindering van gestremdheid gehad, soos bepaal deur die uitgebreide gestremdheid status skaal (EDSS) (p=0,035), terwyl rook MS gestremdheid beduidend verhoog het (p <0.001). Alle SNPs bestudeer was in Hardy-Weinberg ewewig (HWE), met geen beduidende verskille waargeneem in genotipe verspreiding of alleelfrekwensies tussen pasiënte en kontroles nie. Hierdie studie het vir die eeste keer aangetoon dat ‘n diagnose van MS die effek van die FTO rs9939609 polimorfisme op homosisteïen vlakke modereer, wat ooreenstem met die rol van FTO in demetilering en epigenetiese veranderinge. Identifikasie van FTO rs9939609 versterk die belangrikheid van genoegsame folaat inname in die dieet wat akkuraat gemeet kon word deur gebruik te maak van die Mediese Geskiedenis en Leefstyl Vraelys soos toegepas in hierdie studie. Ten slotte, die bevinding dat verhoogde homosisteïen vlakke en BMI statisties betekenisvol beïnvloed word deur leefstylfaktore soos dieet en fisiese aktiwiteit in ons studie populasie, verskaf 'n oplossing om die genetiese bydrae tot hierdie gevestigde vaskulêre risikofaktore vir MS teen te werk. Kombinasie van hierdie inligting met FTO rs9939609 en COMT rs4680 genotipering kan moontlik in die toekoms benut word as deel van 'n omvattende patologie- gesteunende genetiese toetsing strategie wat daarop gemik is om die risikobestuur en kwaliteit van lewe te verbeter in MS pasiënte.

Colorectal cancer is estimated to cause approximately 50,000 deaths each year in the United States. Epidemiological studies have demonstrated an inverse association between sunlight exposure, which stimulates the formation of vitamin D in the skin, and colorectal carcinoma. Laboratory studies report that metabolites of vitamin D, acting through the vitamin D receptor (VDR), regulate cellular proliferation, differentiation and apoptosis. In addition, VDR contains a polymorphic variant, FokI, which results in two different isoforms of VDR. We have demonstrated a differential suppression of β-catenin transcriptional activity by these isoforms in the presence of 1,25(OH)₂D₃ (1,25D). Epidemiological evaluation of metachronous colorectal adenoma formation indicates that VDR includes several single nucleotide polymorphisms (SNPs) which influence the odds of developing colorectal adenoma. In addition, we have found full length Adenomatous Polyposis Coli (APC), a frequently mutated tumor suppressor gene in colorectal cancer, augments both the interaction of VDR and β-catenin as well as the suppression of β-catenin transcriptional activity in the presence of 1,25D. We have also demonstrated in epidemiological studies that the presence of a T-A haplotype in APC codons 486 and 1822, respectively, reduces the odds of any metachronous adenoma by 27% [odds ratio (OR), 0.73; 95% confidence interval (95% CI), 0.59 – 0.91]. Taken together, these data support not only a protective role for vitamin D acting through the VDR, but also for an important role of heritable polymorphic variation in VDR and APC in carcinogenesis.

Asthma is a common complex disease that affects millions of people around the world. Studies indicate the increase in prevalence of asthma worldwide during the past century and report asthma as an important cause of morbidity and mortality. Asthma can be considered as an important health condition in the UK that ranks amongst the countries with the highest rate of asthma prevalence, hospital admissions and mortality due to asthma. Asthma is caused by a combination of genetic and environmental factors. Genetics has an important role in development of asthma with the heritability of around 70% in most studies. To date, more than 100 asthma . associated genes have been identified but they account for only a small proportion of the heritability of asthma. The centerpiece of this thesis is the investigation of genetic association of cystatin and cathepsin genes with asthma and associated phenotypes including atopy and IgE levels. Cathepsinsl cystatins, as proteases and the related antiproteases have been suggested to have a role in airway remodeling. The investigation included three phases; initial association study, replication study in two independent samples sets and complementary analyses. Three sample panels were used in the studies; AUS1/UK1, MRC- AlMRC-E and DLM-4264. The results of this work identified CSTL 1 (cystatin like-1) associated with asthma and IgE levels.