Masters Degrees (Chemical Pathology)

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The association between vitamin D, vitamin D Binding proteins and VDR polymorphisms in diabetic and non-diabetic patients
(Stellenbosch : Stellenbosch University, 2019-03) Maepa, Setjie Welcome; Matsha, Tandi E.; Erasmus, Rajiv T.; Stellenbosch University. Faculty of Medicine and Health Science. Dept. of Pathology. Chemical Pathology.
ENGLISH ABSTRACT: Introduction: Type 2 diabetes mellitus (T2DM) is by far the most prevalent form of diabetes manifesting with insulin resistance (IR), abnormal pancreatic β-cell function and hyperglycaemia. Evidence from epidemiological and observational studies have shown that vitamin D deficiency is associated with increased risk for T2DM although the findings are inconsistent and inconclusive. In the circulation vitamin D is transported bound to vitamin D binding protein (VDBP), evidence showed that vitamin D levels are positively associated with VDBP levels. Several genes such as vitamin D receptor gene (VDR), involved in the metabolic pathway of T2DM have been considered good candidate for susceptibility to T2DM. The present study aimed to investigate the association between vitamin D, vitamin D binding proteins (VDBP) and vitamin D receptor (VDR) polymorphisms in T2DM and non-diabetic patients in the mixed ancestry population. Materials and methods: The current study comprised of 1603 participants (387 males and 1216 females). Vitamin D levels were measured using the paramagnetic particle chemiluminescence test on a Beckman DXI. Vitamin D binding protein (VDBP) in serum samples was measured using the Human Vitamin D BP Quantikine ELISA kit. Fok1 (rs2228570), Apa1 (rs7975232) and Taq1 (rs731236) single nucleotide polymorphisms (SNPs) of the VDR gene were genotyped from a genomic DNA using the TaqMan SNP Genotyping Assays and were confirmed by direct sequencing. Results: Vitamin D deficiency (44%) and insufficiency (42.6%) were highly prevalent and optimal 25(OH)D levels were very low with only 13% having optimal levels. The overall vitamin D status of the whole population group was insufficient (22.0±7.6 ng/mL). 25(OH)D levels and serum VDBP varied according to gender with males having higher 25(OH)D levels (23.6±7 vs 21.5±7.5ng/mL, P=0.0006) and females with significantly higher serum VDBP levels (299.1±71.2 vs 315.9±76.1 µg/mL, P<0.0001). 25(OH)D levels were generally significantly decreased in the hyper-glycemic subgroups. Screen-detected DM males had low 25(OH)D levels compared to normoglycaemic group (17.0±6.1vs 24.2±8.2, P=0.0214). A similar trend was observed in the female groups (21.1±6.0 vs 22.4±7.9, P=0007). Anthropometric measurements including the BMI (kg/m2), Waist C (cm) and Hip C (cm) were significantly higher in hyper-glycaemic group than in normo-glycaemic males and females (All, P<0.0001). In contrast, there were no significant differences in serum VDBP (µg/mL) between the glycaemic sub-groups in either male (P=0.5614) or females (P= 0.4813). The glycaemic parameters, as expected, were significantly increased in the hyperglycaemic sub-groups in both genders, including FBG (mmol/L), 2 hr BG (mmol/L), HbA1c (%), FBI (mIU/L), 2 hr BI (mIU/L) and HOMA-IR (All, both males and females P<0.0001). In general, the lipids, including the triglycerides (mmol/L), LDL-C (mmol/L) and Cholesterol (mmol/L) were also significantly increased in both genders in the hyper-glycaemic sub-groups (All, males P≤0.0300, females P<0.0001), while HDL-C (mmol/L) was significantly decreased in both males and females in the hyperglycaemic sub-groups (All, P≤0.0308). The variant genotype GG of the Fok1, AA of Apa1 and GG of the Taq1 SNPs were not significantly different in hyper-glycaemic patients compared to normo-glycaemic group (58.5% vs 55.1%, P-value, 40.1% vs 38.0%, P-value and 6.9% vs 8.5%, Pvalue,) respectively. Similarly, there was no significant difference in the alleles frequency distribution of these SNPs between the groups. Results also demonstrated no significance difference in the genotype or allele frequency distribution of Fok1 (rs2228570), Apa1 (rs7975232) and Taq1 (rs731236) SNPs between subjects with optimal Vitamin D (25(OH)D ng/mL) levels and those with insufficient/deficient levels (P≥0.2036 and P≥0.6347 respectively). These trends were also observed when serum VDBP levels were evaluated against Fok1, Apa1 and Taq1 genotypes. Multiple linear regression showed that low 25(OH)D was associated with increased LDL-C and PTH in both male and females irrespective of T2DM, but serum VDBP was associated with low 25(OH)D in hyper-glycaemic females only. In normo-glycaemic males 19.5% of the variation in 25(OH)D was attributed to increased LDL-C and in the hyper-glycaemic group 15.5% it was attributed to PTH and CRP. In normo-glycaemic females 12.8% variation in 25(OH)D was attributed to LDL-C, serum creatinine and PTH, whereas in hyper-glycaemic group 16.1% was attributed to increased age, serum VDBP, triglycerides, LDL-C, creatinine and PTH. Conclusion: This study showed prevalence of vitamin D deficiency/insufficiency in the mixed ancestry population group. There was no association between vitamin D (25(OH)D), vitamin D binding proteins (serum VDBP) and VDR polymorphisms in T2DM patients. Serum VDBP levels were associated with low vitamin D levels in hyper-glycaemic females only. Increased LDL-C, PTH and CRP were predictors of low vitamin D levels.
The effect of fructosamine 3 kinase (FN3K) genotypes on the glycation gap in type 2 diabetic and non-diabetic mixed ancestry population of South Africa
(Stellenbosch : Stellenbosch University, 2018-12) Motshwari, Dipuo Dephney; Erasmus, Rajiv T.; Zemlin, Annalise E.; Matsha, Tandi; Stellenbosch University. Faculty of Medicine and Health Science. Dept. of Pathology. Chemical Pathology.
ENGLISH ABSTRACT: Introduction In2017 the International Diabetes Federation (IDF) reported that approximately 425 million adults aged 20-79 years were estimated to have diabetes mellitus (DM) worldwide. The nonenzymatic glycation reactions of proteins such as haemoglobin have been associated with the development of diabetic related complications. These reactions were believed to be irreversible until the discovery of a protein repair enzyme fructosamine 3 kinase (FN3K). This enzyme deglycates glycated haemoglobin (HbA1c) in erythrocytes and other glycated proteins in other tissues. Animal model studies found that the activity of this enzyme varies between individuals leading to differences in HbA1c levels. This results in discrepancies between HbA1c and other glycaemic measures which is termed the glycation gap. The glycation gap is consistent over time within individuals and is associated with diabetic complications. Genetic variants in the FN3K gene have been associated with altered enzyme activity. Therefore, the aim of this study was to examine the role of FN3K genotypes on the glycation gap Methods A total of 1412 subjects (925 normal, 216 pre-diabetic and 271 type 2 diabetics), with 339 males and 1073 females aged ≥ 20 years of mixed ancestry descent, residing in Bellville South, South Africa were included in this study. The diabetics were diagnosed using the oral glucose tolerance test. The glycation gap was determined according to a formula: Glycation gap= HbA1c - FHbA1c, (FHbA1c = {[(fructosamine- mean fructosamine)/SD fructosamine] X SD HbA1c} + mean HbA1c). DNA was extracted from whole blood using the salt extraction method. FN3K single nucleotide polymorphisms (SNPs) were genotyped with the Applied Biosystems™ QuantStudio™ 7 Flex Real-Time PCR System 96 well fast from Thermo Fisher Scientific. HbA1c was measured using HPLC (Biorad Variant Turbo) and fructosamine was measured using a colorimetric test nitro-blue-tetrazolium (NBT). Results SNP c. -232A/T deviated from Hardy Weinberg Equilibrium (HWE) and was left out for the rest of the statistical analysis. The polymorphism G900C followed the Hardy-Weinberg Equilibrium and was therefore studied. The genotype frequencies for SNP G900C in the glycaemic sub-groups were as follows, GG: 45.9 %, GC: 43.7 %, CC: 10.4 % in normal subjects; GG: 48.6 %, GC: 41.7 %, CC: 9.7% in pre-diabetics and GG: 41.7 %, GC: 46.5 %, CC: 11.8 % in diabetics, and they followed the Hardy-Weinberg equilibrium. There were no significant differences in the SNP G900C genotype frequencies between the glycaemic subgroups. The glycation gap significantly decreased across the GG, GC and CC genotype variants in males, mean ± SD were -0.13±0.86, -0.25±0.72 and -0.80±1.04 respectively, (P=0.0239). However the difference was not observed in females. Moreover the glycation gap showed a positive correlation with non glycaemic factors including body mass index (BMI) (r=0.3694, p<0.0001), waist circumference (waistC) (r=0.3749, p<0.0001), hip circumference (hipC) (r0.3151, p<0.0001), triglycerides (r=0.2540, p<0.0001) and a negative correlation with high density lipoprotein cholesterol (HDL-Chol) (r=-0.2031, p<0.0001). Conclusion In conclusion the present study found that the glycation gap might be influenced by genetic In conclusion the present study found that the glycation gap might be influenced by genetic active mechanisms in the intracellular erythrocyte compartment. Identification of the G900C polymorphism in an early stage of diabetes could be useful especially in therapeutic decisions and prediction of improved prognosis. However, there are other confounding factors influencing the glycation gap and future studies are required to confirm these findings.
Screening for Chronic Kidney Disease (CKD) in a high risk population using a Point of Care Instrument for creatinine measurement: A community based study (The Bellville South Africa Study)
(Stellenbosch : Stellenbosch University, 2017-12) Krige, Tammy; Erasmus, Rajiv T.; Rensburg, Megan; Stellenbosch University. Faculty of Medicine and Health Science. Dept. of Pathology. Chemical Pathology.
ENGLISH ABSTRACT : Chronic kidney disease (CKD) is described as abnormal kidney function in which one third is lost over a period of 3 months and is a global epidemic with a particularly concentrated incidence within developing countries, such as Sub-Sahara Africa (SSA). Health facilities in SSA are limited due to lack of funding and a dearth in disease and medical knowledge. This coupled with the high incidence of both communicable and non-communicable diseases makes for an ideal environment for the implementation of Point-of-Care Testing (POCT), defined as an analytical test that is performed near the patient, delivering results in real time without the need for a conventional laboratory. CKD POCT involves the measurement of creatinine in capillary whole blood samples in order to determine the estimated glomerular filtration rate (eGFR) of patients in order to stage their CKD status from stage 1-6. This study aimed to bridge the gap in knowledge with regard to cut-offs of creatinine levels and eGFR values when screening a mixed ancestry populations. Currently there is only documented and standardized cut-offs for Caucasian and African American populations. This study looked at the African mixed ancestry population and acts as a starting point for standardizing POCT cut-offs for other international mixed ancestry populations. 103 participants were recruited from the Bellville South community, Cape Town, South Africa. The study was a comparative study that was designed to evaluate the Nova Statsensor® point of care instrument for the measurement of creatinine for the detection of CKD in adult mixed ancestry subjects from the Bellville South Community in South Africa. Secondary objectives included (1) the prevalence of CKD based on the results of the instrument, and (2) the correlation between the Nova Statsensor®, and the central laboratory creatinine values (IDMS traceable). Ancillary objectives of the study were to evaluate the technical quality of POC testing for creatinine in a community setting, as well as the evaluation of the cost implications when introducing this form of POCT into a primary care setting. The study found that the Nova Statsensor® in this study had a sensitivity of 66.7% and a specificity of 100%, displaying excellent diagnostic accuracy. It was found that the device displayed negative proportional bias which may lead to future CKD patients being misdiagnosed as healthy within screening programmes. The prevalence was found to be 2.9% within this mixed ancestry population. The device was user friendly and requires a small sample volume, however it is costly to implement. The laboratory evaluation study found that the Nova Statsensor® creatinine meter produced a direct creatinine concentration comparison that was less than expected, possibly due to creatinine levels depending on several factors which include muscle mass, obesity, gender, and age and having a wide reference interval. Thus highlighting the importance of the use of the equations to calculate eGFR in CKD screening in order to obtain the CKD staging results which displayed better correlation to the reference method, compared to creatinine measurement alone. The device was comparable to the reference method when performance was measured based on CKD staging through the calculation of the MDRD equation.
E-Selectin, and markers of HIV disease severity, inflammation and coagulation in treatment- naïve individuals living with HIV
(Stellenbosch : Stellenbosch University, 2015-12) Hoffman, Madelein; Zemlin, A. E.; Ipp, H.; Erasmus, R. T.
ENGLISH SUMMARY: Background: E-selectin is an adhesion molecule that is expressed on the surface of activated endothelial cells. During inflammation the endothelial cells are activated, trafficking cells of the immune system through the endothelial wall to the point of inflammation. Human Immunodeficiency Virus (HIV) infection causes continuous and long term activation of the immune system and has an increased incidence of cardiovascular disease. Selectins play an important role in atherosclerotic plaque formation as continuous activation leads to plaque formation and eventual plaque rupture with subsequent thrombosis and the initiation of a cardiac event. The aim of this study was to determine the levels of E-selectin in an HIV infected and control population and to correlate these levels with markers of HIV disease severity, inflammation and coagulation in anti-retroviral treatment (ART)-naïve HIV infected individuals. Methods: E-selectin levels were determined using ELISA in 180 participants from an HIV prevention and testing clinic in Crossroads, Cape Town. There were 114 HIV infected cases and 66 HIV negative controls. These levels were compared with each other and correlated to various other markers associated with HIV disease severity (viral load and CD4+count), inflammation (white cell count (WCC), high sensitivity C-reactive protein (hsCRP), %CD38/8, albumin and IgG) and coagulation (fibrinogen and D-dimer). Results: A total of 75% of the females tested positive for HIV compared to 37% of the males. Statistics comparing HIV status with WCC, CD4+count, %CD38/8, albumin, IgG, hsCRP and D-dimer found significant differences (p<0.01) between the two groups. No differences in E-selectin (p=0.84) and fibrinogen (p=0.65) levels were found between the cases and the controls. When E-selectin was compared with all the analytes tested, significant correlations were found with age (p=0.02) and gender (p=0.01). Albumin (p=0.05) showed a significant correlation with E-selectin in the control group. The correlation with the WCC (p=0.07) in the HIV infected group neared significance. Conclusion: No significant difference in E-selectin levels was found between the HIV positive and negative control group and no correlations were found with Eselectin and the markers of disease severity, inflammation and coagulation. Thus we found E-selectin to be a poor marker of inflammation in this setting. As age and gender are established markers of CVD and males have higher E-selectin levels than females, the lack of significance may be due to our sample population’s young age (mean 31 years) or the fact that 70% of the cohort was female. Thus significant endothelial damage may not yet have taken place to increase E-selectin levels. In addition, this HIV group was predominantly in the chronic stage of infection, therefore the increase in E-selectin levels may have occurred earlier during the acute infection.
The 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.

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