Doctoral Degrees (Chemical Pathology)

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Browsing Doctoral Degrees (Chemical Pathology) by browse.metadata.advisor "Kotze, Maritha J."

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    Investigation of the role of vitamin D metabolism in South African breast cancer patients using a pathology-supported genetic testing platform
    (Stellenbosch : Stellenbosch University., 2020-03) Okunola, Abisola Oyedele; Kotze, Maritha J.; Erasmus, Rajiv T.; Zemlin, Annalise E.; Torrorey-Sawe, Rispah; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Chemical Pathology.
    ENGLISH ABSTRACT: The high global breast cancer incidence drives the development of novel genomic approaches for disease prevention and targeted treatment. Towards this goal, a pathology-supported genetic testing (PSGT) platform was established to facilitate risk management of non-communicable diseases across the continuum of care, ranging from early-stage to metastatic disease and cancer survivorship. The causes and consequences of low vitamin D levels recently reported in the majority of postmenopausal breast cancer patients treated with aromatase inhibitors at the Tygerberg Academic Hospital, in the Western Cape Province of South Africa, were addressed in this study using PSGT to translate genomic findings into clinical practice. The aim was to determine the relationship between clinical characteristics, tumour histopathology and genetic variation underlying vitamin D metabolism in postmenopausal breast cancer patients at increased risk of osteoporosis, identified as a significant co-morbidity in the study population. Clinical and lifestyle information of 116 postmenopausal women with known vitamin D status diagnosed with breast carcinoma between 2014 and 2017 was extracted from a central genomics database linked to a biobank of DNA samples extracted from blood. Whole exome sequencing (WES) was performed on the Ion Torrent platform, followed by variant calling of vitamin D-related genes, while simultaneously assessing BRCA1/2 mutation status. Allele-specific real-time polymerase chain reaction (PCR), Sanger sequencing and long-range nanopore sequencing using the pocket-size MinION device were used to verify the WES results and to screen for variants in the vitamin D receptor (VDR) and E-cadherin (CDH1) genes beyond the coding regions covered by WES. Seasonal variation (p = 0.009) and high body mass index (BMI) (p = 0.032) contributed significantly to vitamin D levels, with the lowest values recorded during winter. WES initially performed in 10 breast cancer patients selected based on vitamin D levels at extreme upper and lower ranges, identified GC rs4588 (c.1364C>A, T455K) as a potential contributing factor vitamin D deficiency in the five patients with ultra-low vitamin D levels (≤12 ng/mL). However, 2/5 patients with levels in the upper extreme of vitamin D (>30 ng/mL), also tested positive for this variant and no significant association was detected after extended genotyping in 100 South African patients using real-time PCR. Sanger sequencing subsequently performed in 14 breast cancer patients diagnosed with osteoporosis prior to initiation of aromatase inhibitor therapy, highlighted the potential significance of genetic variation in the VDR gene. WES analysis of VDR in an extended sample of 55 breast cancer patients furthermore confirmed the significant effect of genetic variation in this gene on bone health (p < 0.001). The CDH1 gene known to be activated by VDR was furthermore analysed in patients stratified by tumour type. CDH1 c.G671A (p.R224H) detected in a breast cancer patient with invasive carcinoma of no special type (ICNST) was classified as benign, since pathogenic germline CDH1 variants are associated with invasive lobular carcinoma and diffuse gastric cancer, but not ICNST. CDH1 c.A1298G (p.D433G) was detected in a patient with invasive lobular carcinoma together with a pathogenic BRCA1 variant detected by WES. Although this finding supports a likely benign classification for CDH1 p.D433G as reported in the international ClinVar database, a family history of stomach cancer raised the possibility of a CDH1 modifier gene effect on BRCA1 gene expression. New insights gained through integration of pathology and genomic findings were incorporated into a pharmaco-diagnostic algorithm applicable to hormone receptor-positive postmenopausal breast cancer patients. The PSGT platform facilitated interpretation of research results of study participants through use of WES and recommendation of genetic counselling where appropriate.
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    Molecular investigation of genetic factors associated with insulin resistance and obesity in a South African population
    (Stellenbosch : Stellenbosh University, 2015-12) Vergotine, Zelda; Erasmus, Rajiv T.; Matsha, Tandi E.; Pillay, Tahir S.; Kotze, Maritha J.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Chemical Pathology.
    ENGLISH ABSTRACT: Background: The aetiopathogenesis of type 2 diabetes and the associated insulin resistance have been shown to have a strong genetic basis. Several genetic variants of the peroxisome proliferatoractivated receptor gamma (PPARG) and the insulin receptor substrate (IRS) 1 genes have been associated with the metabolic states of obesity, insulin resistance and type 2 diabetes in Caucasian populations. Furthermore, insulin resistance is strongly associated with diabetes and subsequent cardiovascular disease. These are increasingly common in low- to middle -income countries, including South Africa. Limited information is currently available regarding genetic associations with insulin resistance in African populations. Objectives: (1) To identify subjects with insulin resistance and determine the frequencies of the single nucleotide polymorphisms in the PPARG and IRS1 genes and examine the associated risk of insulin resistance and type 2 diabetes mellitus in a mixed-ancestry South African population. (2) To investigate the relationship between indices of insulin resistance and carotid intima media thickness, a marker of subclinical cardiovascular disease/atherosclerosis. Methods: A total of 856 (235 males) mixed-ancestry adults drawn from an urban community of Bellville South, Cape Town were genotyped for PPARG Pro12Ala (rs1801282, G>C), Pro115Gln (rs1800571, G>T), Val290Met (rs72551362, G>A), Pheu388Leu (rs72551363, T>A), Arg397Cys (rs72551364, C>T), His449His (rs3856806, C>T) and IRS1 Gly972Arg (rs 1801278, G>A). The oral glucose tolerance test was performed and cardiometabolic risk factors measured. Insulin resistance was estimated by the homeostasis model assessment of insulin resistance, the homeostasis model assessment of functional beta-cells, the quantitative insulin-sensitivity check index, the fasting insulin resistance index and the glucose/insulin ratio. Carotid intima media thickness was measured in longitudinal section at the far wall of the distal common carotid arteries, 2 cm from the bifurcation, at three consecutive end-points, 5-10 mm apart. Results: The genotype frequencies of PPARG Pro12Ala, IRS1 Gly972Arg and PPARG His449His were 10,4%, 7,7% and 23,8% respectively. No mutations were found for PPARG Pro115Gln, Val290Met, Pheu388Leu and Arg379Cys. In a model containing both PPARG Pro12Ala and IRS1 Gly972Arg alleles and their interaction term, the presence of the PPARG Pro12 resulted in a 64% risk of prevalent type 2 diabetes mellitus and was associated with higher 2 hour post-OGTT insulin levels in subjects with normoglycaemia. The PPARG Pro12 was associated with insulin resistance and interacted with IRS1 Gly972Arg, increasing the risk of type 2 diabetes mellitus. The PPARG His449His allele T frequency was about 14% and in an additive genetic model significantly reduced the risk of diabetes by 44%. After adjustment for age, gender, body mass index and diabetes status, the fasting plasma glucose (β=0,087;p=0,042) and glucose/insulin ratio (β=0,026; p=0,026) were associated with carotid intima media thickness. However, the effect on the overall model performance was marginal, R2<29,7%. Conclusion: The PPARG Pro12 was associated with insulin resistance and showed a gene-gene interaction with the unfavorable polymorphism IRS1 Gly972Arg, leading to an increased risk of type 2 diabetes mellitus. In contrast, the PPARG His449His T allele showed a protective effect against the risk of developing diabetes. Furthermore, indices of insulin resistance such as homeostatis model assessment of insulin resistance, quantitative insulin-sensitivity check index, fasting insulin resistance index and the glucose/insulin ratio were weakly associated with carotid intima media thickness in the risk stratification of cardiovascular disease in this population.
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    Mutational analysis of the solute carrier family 11 member 1 gene (SLC11A1) implicated in iron transport
    (Stellenbosch : Stellenbosch University, 2003-12) Zaahl, Monique G. (Monique Glenda); Kotze, Maritha J.; Warnich, L.; Winter, T. A.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences.
    ENGLISH ABSTRACT: The solute carrier family 11 member 1 gene (SLC11A 1) is a divalent metal ion transporter with various pleiotropic effects on macrophage function. This gene that regulates iron, and is also regulated by cellular iron levels, has previously been linked to many infectious and autoimmune diseases. In this analysis, in vitro studies using the luciferase reporter system as well as case-control association studies were applied to investigate the significance of SLC11 A1 allelic variation in patients with diverse disease phenotypes. For in vitro studies, five different SLC11A 1 promoter constructs were generated, followed by transfection into U937 and THP-1 cells. The inserted fragments included two previously described alleles (alleles 2 and 3), two novel alleles identified in this study (alleles 8 and 9) and a C to T point mutation at nucleotide position -237 in the presence of allele 3. The most striking finding was the opposite effect observed for allele 3 in the presence of the -237C~ T polymorphism, similar to that of allele 2. Although the SLC11A 1 gene has previously been implicated in iron transport, we have demonstrated, for the first time, that the various alleles investigated cause differential expression of the gene upon iron loading. Association studies were performed by investigating diseases including oesophageal cancer (DC), inflammatory bowel disease (lBO) and hereditary haemochromatosis (HH) (or primary iron overload). Significant associations (P