Masters Degrees (Chemical Pathology)
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Browsing Masters Degrees (Chemical Pathology) by Subject "Dissertations -- Medicine"
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- ItemAnalysis of single nucleotide polymorphisms with opposite effects on serum iron parameters in South African patients with multiple sclerosis(Stellenbosch : Stellenbosch University, 2014-04) Moremi, Kelebogile Elizabeth; Van Rensburg, S. J.; Kotze, Maritha J.; Geiger, D.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology, Chemical Pathology.ENGLISH ABSTRACT: There is growing interest in how genetic and environmental risk factors interact to confer risk for dysregulated iron homeostasis, which is considered a possible pathogenic mechanism in multiple sclerosis (MS). While iron deficiency has been associated with greater disability and disease progression, cerebral accumulation and overload of insoluble iron has also been reported in MS patients. Variation in the matriptase-2 (TMPRSS6) gene has recently been described that may lead to reduced iron levels, which raised the question of whether it may be involved in dysfunctional iron regulation as a pathogenic mechanism in MS. The aims of the study were as follows: 1)) comparison of the allele frequencies and genotype distribution for TMPRSS6 A736V (rs855791, c.2207C>T) and HFE C282Y (rs1800562, c.845G>A) between patients diagnosed with MS and unaffected controls; 2) determination of the effects of clinical characteristics, relevant lifestyle factors and genotype on serum iron parameters in MS patients compared to population matched controls; and 3) determination of clinical outcome in relation to age of onset and degree of disability in MS patients. The study population included 121 Caucasian MS patients and 286 population-matched controls. Serum iron, transferrin, ferritin and transferrin saturation levels were available from previous studies and lifestyle factors were subsequently documented in a subgroup of 68 MS patients and 143 controls using the study questionnaire. Genotyping of TMPRSS6 A736V and HFE C282Y were performed using allele-specific TaqMan technology. The genotype distribution and allele frequencies of TMPRSS6 A736V and HFE C282Y did not differ between MS patients and controls. MS patients homozygous for the iron-lowering minor T-allele of TMPRSS6 A736V had significantly lower serum iron levels (p=0.03) and transferrin saturation levels (p=0.03) compared to CC homozygotes. In MS patients the iron-loading minor A-allele of HFE C282Y was also associated with a paradoxical decrease in serum ferritin (p<0.01) compared to GG homozygotes. When considering the combined effect of the minor alleles of TMPRSS6 A736V and HFE C282Y with opposite effects on iron levels, we found a significant reduction in serum ferritin levels (p<0.05), independent of age, sex, body mass index (BMI) or dietary red meat intake in MS patients. A similar effect was not observed in the population- and age-matched controls. Higher dietary red meat intake correlated significantly with increased ferritin only in controls (p=0.01 vs. 0.21 for MS patients). In the presence of the minor allele of HFE C282Y, the TMPRSS6 A736V CT and TT genotypes were associated with a significantly earlier age of onset of MS when the post hoc test was applied (p=0.04). All the study aims were successfully accomplished. Our results support the possibility of an epistatic effect between TMPRSS6 A736V and HFE C282Y associated with reduced ferritin levels in MS patients. Pathology-supported genetic testing (PSGT) applied in this study as a new concept for analysis of complex diseases with a genetic component, is well placed to optimise clinical management in patients with MS.
- ItemThe development and application of a polymerase chain reaction (PCR) based assay to determine the impact of genetic variation in South African patients diagnosed with depression(Stellenbosch : Stellenbosch University, 2014-04) Delport, Darnielle; Schoeman, R.; Kotze, Maritha J.; Geiger, D.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology, Division of Chemical Pathology.ENGLISH ABSTRACT: Major Depressive Disorder (MDD) is a severe debilitating medical condition that may lead to suicide. Due to a poor understanding of the biological mechanisms underlying the disease process therapeutic decisions are usually taken using a ‘trial and error’ approach. This is not ideal since many treatments do not work as expected for all individuals. Studies have shown that only half of MDD patients receive the appropriate treatment, whereas many patients have adverse response to anti-depressants. These may include weight gain and raised homocysteine levels that may further compromise the health status of MDD patients and may partly explain the link with cardiovascular disease. The objective of the study was to identify genetic risk factors interacting with environmental factors implicated in MDD that may be of relevance to the South African population. Polymorphisms in the MTHFR (677 C>T, rs1801133 and 1298 A>C, rs1801131), COMT (472G>A, rs4680), CYP2D6 (6937G>A, rs3892097), ASMT (24436 G>A, rs4446909) and SLC6A4 (43 bp ins/del, rs4795541) genes were genotyped in 86 MDD patients and 97 population-matched controls. The specific aims were 1) to analytically validate high throughput real-time polymerase chain reaction (RT-PCR) genotyping assays for the 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 MDD. A total of 183 unrelated Caucasians participated in the study, including 69 females and 17 males with MDD and 57 female and 40 male controls without a personal and family medical history of overlapping stress/anxiety and depressive disorders. All study participants were genotyped for the six selected SNPs considered clinically useful based on international data. The allelic distribution of the SNPs, single or combined into a genotype risk score after counting their minor alleles, did not differ between MDD patients and controls. Homocysteine levels were determined and correlated with body mass index (BMI) and other variables known to influence these phenotypes. The folate score assessed with use of the study questionnaire was significantly lower in the patient group compared with controls (p=0.003) and correlated significantly with BMI, particularly in females (p=0.009). BMI was on average 8% higher in the MDD patients compared with controls (p=0.015) after adjustment for age and sex. The MTHFR rs1801133 677 T-allele was associated with a 14% increase in BMI in MDD patients but not controls (p=0.032), which in turn was associated with significantly increased homocysteine levels (p<0.05). The aims of the study were successfully achieved. Identification of the MTHFR rs1801133 677 T-allele reinforces the importance of adequate folate intake in the diet due to increased risk of obesity and depression found to be associated with low dietary intake. Evidence of shared genetic vulnerability for many chronic diseases and drug response mediated by the MTHFR 677 T-allele support the clinical relevance of this low-penetrance mutation.
- ItemDevelopment of a pathology-supported genetic test for improved clinical management of patients diagnosed with multiple sclerosis(Stellenbosch : University of Stellenbosch, 2010-12) Jalali Sefid Dashti, Mahjoubeh; Janse van Rensburg, Susan; Kotze, Maritha J.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Chemical Pathology.ENGLISH ABSTRACT: The aetiology of multiple sclerosis (MS) remains largely unknown, due to its multifactorial nature with environmental and genetic factors contributing to the risk. Several investigations highlighted the important role of the genetic component influencing disease susceptibility and progression. In the present study genetic variations in the MTHFR (1298 A>C and 677 C>T) and HFE (845 G>A) genes previously, shown to affect folate and iron metabolism respectively, were studied in the context of MS. The aim of the study was to contribute the laboratory component of a pathology supported genetic testing approach used to identify a subgroup of MS patients with altered nutritional requirements due to genetic susceptibilities. The study population included 90 patients with a clinical diagnosis of MS and 49 control individuals, without any signs or symptoms of the disease, drawn from the same age- and population group. Three mutation detection systems were compared in terms of accuracy, sensitivity, cost effectiveness and ease of operation in relation to the MTHFR and HFE gene mutations analysed. Analytical validity of the genetic assays was an important consideration; therefore the respective real-time polymerase chain reaction (RT-PCR) methods were compared with direct DNA sequencing as the gold standard. The methodology included use of the ABI™ 7900HT, the Roche LightCycler® 480 II system and the Corbett Rotor-Gene™ 6000 5-plex HRM. The same genotype results were obtained for the DNA samples tested with the three RT-PCR methods. In terms of cost effectiveness, ease of operation and optimization, the Corbett Rotor-Gene™ 6000 5-plex HRM thermal cycler, with use of the ABI™ TaqMan Genotyping assays was found to be the most efficient for mutation detection using relatively small sample batches. Following successful standardization of the RT-PCR assays, genotype-phenotype correlation studies was performed in a subset of 43 MS patients with available data. Biochemical tests were previously done on blood samples at the National Health Laboratory Service (NHLS) chemical pathology laboratory at Tygerberg Academic Hospital. A novel finding of this study was that heterozygotes and homozygotes for mutation 1298 A>C in the MTHFR gene presented with lower serum iron levels (12.37 ± 5.91 μmol/l) in comparison to subjects without the C-allele (18.64 ± 7.15 μmol/l; P = 0.02). Furthermore, C-reactive protein (CRP) levels were found to be marginally significantly higher (P = 0.07) in the MTHFR 1298 A>C mutation-positive heterozygotes compared to subjects without the C-allele (6.65 ± 4.96 mg/l vs 2.93 ± 2.31 mg/l), linking inflammation to the presence of the MTHFR 1298 A>C mutation. In comparison, the MTHFR 677 C>T as well as the HFE 845 G>A mutation showed no correlation with transferrin saturation, ferritin, haemoglobin or CRP levels. The absence of increased iron status in HFE mutation carriers was in accordance previous findings suggesting altered iron metabolism in MS patients with this mutation. For the first time, high-throughput assays for functional polymorphisms in the MTHFR and HFE genes can now be offered as a routine service at the Tygerberg Academic Hospital. This application is used in combination with blood biochemistry tests as part of a comprehensive gene-based, pathology supported screening and intervention program aimed at improved quality of life in patients diagnosed with MS.
- ItemInvestigation of the relationship between genetic and environmental risk factors associated with obesity and insulin resistance in South African patients with non-alcoholic fatty liver disease(NAFLD)(Stellenbosch : Stellenbosch University, 2012-12) Pretorius, Jakobus; Kotze, Maritha J.; Hoffmann, Mariza; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Chemical Pathology.ENGLISH ABSTRACT: Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease in the world. The disease spectrum of NAFLD extends from steatosis (types 1,2) to non-alcoholic steatohepatitis (NASH) with inflammation (types 3,4). The aims of the study were 1) to analytically validate high-throughput real time polymerase chain reaction (RT-PCR) assays for three selected single nucleotide polymorphisms (SNPs), FTO rs9939609 (intron 1 T>A), TNF-α rs1800629 (-308 G>A) and PPARγ rs1801282 (Pro12Ala, 34 C>G), and 2) to perform genotype-phenotype association studies in relation to biochemical abnormalities, disease severity and age of onset. A total of 119 patients with fatty liver identified on ultrasound, including 88 histologically confirmed NAFLD patients, and 166 control individuals were genotyped for the three selected SNPs. RT-PCR validated against direct sequencing as the gold standard was used for detection of genetic variation. All three SNPs were in Hardy Weinberg equilibrium in the study population, except for a deviation in genotype distribution detected for PPARγ rs1801282 in the NAFLD patient subgroup (p<0.001). After adjustment for age and gender, the risk-associated FTO rs9939609 A-allele was detected at a significantly higher frequency in the Caucasian compared with Coloured patients (p=0.005). The opposite was detected for the risk-associated TNF-α rs1800629 A-allele, which occurred at a significantly higher frequency in the Coloured compared with Caucasian NAFLD patients (p=0.034). The onset of fatty liver disease symptoms was on average 5 years younger in the presence of each risk-associated TNF-α rs1800629 A-allele (p=0.028). When considered in the context of an inferred genotype risk score ranging from 0-6, disease onset occurred on average 3 years earlier (p=0.008) in the presence of each risk-associated FTO A-allele, TNF-α A-allele or PPARγ C-allele. After adjustment for age, gender and race, no differences in genotype distribution or allele frequencies were observed between histologically confirmed NAFLD (types 1,2) and NASH (types 3,4) patients, while the minor allele frequency for the TNF-α rs1800629 was significantly higher in the total NAFLD (types 1-4) (p=0.047) as well as NASH subgroup (NAFLD types 3,4) (p=0.030) compared with obese patients without a histologically confirmed NAFLD diagnosis. A significant correlation was furthermore observed between the number of TNF-α rs1800629 A-alleles and increasing CRP levels (p=0.029), with a favourable reduced effect in the presence of low- to moderate alcohol intake. The average waist circumference of physically active NAFLD patients was 12% lower than in physically inactive patients (p=0.004). In view of the results presented in this study, the inclusion of the selected SNPs, and in particular the pro-inflammatory TNF-α rs1800629 polymorphism, may be considered as part of a comprehensive cardiovascular risk evaluation of NAFLD patients. Ultimately, early detection of patients with fatty liver disease symptoms and effective intervention based on the underlying disease mechanism may prevent progression from NAFLD to NASH, shown to be an independent risk factor for cardiovascular diseases.
- ItemLipoprotein X : biochemical predictors and detection by non-denaturing polyacrylamide gradient gel electrophoresis(Stellenbosch : Stellenbosch University, 2007-12) Le Riche, Mia; Marias, David; Erasmus, Rajiv T.; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Chemical Pathology.ENGLISH ABSTRACT: Lipoprotein X (LpX) is an abnormal cholesterol-containing particle that may be present in the serum of subjects with cholestasis, lecithin:cholesterol acyltransferase (LCAT) deficiency and parenteral nutrition. The biochemistry, metabolism, clinical significance and laboratory analysis of LpX is discussed in this study. This laboratory-based project investigated icteric samples received at the Chemical Pathology laboratory, Tygerberg Hospital, for serum predictors of LpX and the use of a modified non-denaturing polyacrylamide gradient gel electrophoresis system in the detection of LpX. The study showed that the non-denaturing polyacrylamide gradient gel electrophoresis system (2-8%) is a useful test in demonstrating LpX in icteric plasma and has potential for a screening test in LCAT deficiency. Serum concentration of conjugated bilirubin, alkaline phosphatase, gamma glutamyltransferase, free cholesterol, phospholipid, free cholesterol: total cholesterol ratio and conjugated bilirubin: total bilirubin ratio are all good predictors of LpX. The ratio of free cholesterol to total cholesterol (FC/TC > 0.6) was the best predictor of LpX. In the setting of obstructive liver disease LpX is seen in 66% of patients if total cholesterol is > 7.5 mmol/L.
- ItemThe occurance of genetic variations in the MYH9 gene and their association with CKD in a mixed South African population(Stellenbosch : Stellenbosch University, 2012-12) Masconi, Katya; Matsha, Tandi; Erasmus, Rajiv T.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology.ENGLISH ABSTRACT: The purpose of this study was to investigate the association of the selected MYH9 single nucleotide polymorphisms (SNPs) with chronic kidney disease (CKD) and its related co-morbidities in the South African mixed ancestry population residing in Bellville South, Cape Town. In 2008, two landmark studies identified SNPs in the MYH9 gene which explained most of the increased risk for non-diabetic CKD in African Americans. These polymorphisms were later found to be weakly associated with diabetic nephropathy. Three SNPs that exhibited independent evidence for association with CKD were selected (rs5756152, rs4821480 and rs12107). These were genotyped using a Taqman genotyping assay on a BioRad MiniOpticon and confirmed by sequencing in 724 subjects from Bellville South, Cape Town, South Africa. Prevalent CKD was defined based on the estimated glomerular filtration rate calculated using the modification of diet in renal disease (MDRD) formula. Chronic kidney disease was present in 214 subjects (29.6%), 96.3% were stage 3 and only 8 subjects were stage 4. In additive allelic models, adjusted for age and gender, rs5756152 demonstrated an association with kidney function whereby each G allele of rs5756152 increased eGFR by 3.67 ml/min/1.73, reduced serum creatinine by 4.5% and increased fasting plasma glucose by 0.51 mmol/L. When an interaction model was used, the effect of rs5756152 on serum creatinine, eGFR and blood glucose levels was retained, and enhanced, but only in diabetic subjects. In addition, rs4821480 T allele increased eGFR while rs12107 A allele decreased glucose levels in diabetic subjects. In contrast to reports that MYH9 SNPs are strongly associated with non-diabetic end stage renal disease, our study demonstrated that rs5756152 and rs4821480 are associated with early kidney function derangements in type 2 diabetes whilst rs12107 is associated with glucose metabolism. Our findings, along with previous reports, suggest that the MYH9 gene may have a broader genetic risk effect on different types of kidney diseases than previously thought.