Masters Degrees (Anatomical Pathology)

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Analysis of hereditary haemochromatosis and clinical correlations in the elderly
(Stellenbosch : Stellenbosch University, 2000-12) Bouwens, C. S. H.; Kotze, Maritha J.; Maritz, F. J.; De Villiers, J. N. P.; Stellenbosch University. Faculty of Medicine & Health Sciences. Dept. of Pathology.
ENGLISH ABSTRACT: Hereditary haemochromatosis (HH) is an autosomal recessive iron storage disease where the accumulation of iron in parenchymal organs may lead to diabetes, heart failure, liver cirrhosis, arthropathy, weakness and a variety of other ailments if preventive measures are not taken. HH is often not considered as a cause of these conditions, particularly not in the elderly where the background frequencies of type II diabetes, osteoarthritis and heart failure are generally high. Heterozygosity for C282Y, the HFE-mutation causing HH in approximately 80% of affected individuals worldwide, has been linked to a raised incidence of malignancies of the colon and rectum, stomach and the haematological system. One of the highest carrier-frequencies (116) in the world for this mutation has been reported in the South-African Afrikaner population, resulting in C282Y-homozygosity in approximately 1 in every 115 people in this group. A sample of 197 elderly Afrikaner volunteers was recruited for genotype/phenotype association studies. Their clinical presentation was denoted, biochemical iron-status determined and HFE genotyping performed. Either an increase or decrease in survival, or both, were proposed, depending on possible gender effects. HH has been positively associated with various cancer types, but may also protect against iron-deficiency anaemia which is by far the most frequent cause of anaemia in the older person. This study has led to the following findings: 1. The carrier frequency of mutation C282Y was found to be 1/8 in the elderly population (similar in males and females), which is slightly lower than the 1/6 reported in younger adults from the same population. Only one C282Y homozygote and two C282YIH63D compound heterozygotes were detected, all of them female. 2. The prevalence of diabetes, heart disease, arthropathy or a combination of these conditions did not differ significantly in C282Y heterozygotes and the mutationnegative group. 3. Among 24 C282Y heterozygotes only one individual with rectal carcmoma was detected compared with two cases with rectal- and seven with colonic malignancies in 153 mutation-negative individuals. The single female C282Y homozygote identified suffered from both rectal and colon carcinoma and died approximately 6 months ago as a consequence of her colon malignancy. 4. Serum ferritin appears to be a highly unreliable parameter of iron status, particularly in the elderly where a variety of factors that may influence the levels are often present in elderly individuals. This may be due to ageing alone or as a result of multiple comorbidities. 5. Serum ferritin levels were lower than expected in elderly subjects with mutation C282Y and compound heterozygotes with both C282Y and H63D, which may be related to a variable penetrance of the HFE gene mutations. It is possible that variation in other genes exist that confer protection against iron-loading by gene-gene interaction. The probability that environmental factors (e.g. a low iron diet) are more important in this respect cannot be excluded, although this is considered less likely in the light of the fact that the same trend was observed in all mutation-positive elderly individuals. It is therefore highly likely that C282Y -positive subjects with significant iron loading have died before reaching their seventies, particularly since none of the males included in this study were homozygous or compound heterozygous for the mutations analysed. In conclusion, possession of a mutant HFE gene does not appear to confer a survival advantage in old age, neither does it seem that mutation carriers with significant ironloading are overlooked by the medical fraternity. Further investigations are warranted to shed more light on the contributions of gene-gene and gene-environment interaction in the clinical manifestation of Hll, and how these processes can be manipulated to prevent the symptoms of this largely underdiagnosed disease.
Comparison of Xpert® Breast cancer STRAT4 assay and immunohistochemistry for the evaluation of breast cancer biomarkers in South African patients
(Stellenbosch : Stellenbosch University, 2020-12) Dube, Welile Vumile; De Jager, Louis; Kotze, Maritha J.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Anatomical Pathology.
Background: Breast cancer is one of the most common cancers diagnosed in women and approximately 60% of breast cancer related deaths are reported in low-and middle-income countries. Breast cancer is a highly heterogeneousdisease, and molecular subtyping is paramount foreffective treatment of patients. Therefore, it is important to validate new molecular methods for assessing cancer biomarkers for cost-effective use in resource-poor settings.Aim:Aretrospective study was performed to determine the concordance between aQuantitativeReverse TranscriptionPolymerase Chain Reaction(RT-qPCR) CE-IVD assay (Xpert® Breast Cancer STRAT4*) and the current gold standard methods of immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for determining estrogenreceptor (ER),progesterone receptor(PR), human epidermal growth factor receptor 2 (HER2)andproliferation index(KI-67)expression in breast carcinomas.Method: One hundred and one cases of breast carcinoma were retrieved from the archives of the Division of Anatomical Pathology, Tygerberg Academic Hospital. The original stained slides were reviewed and IHC expression of ER, PR, HER2 and KI-67 scored. Three-micron sections were cut from formalin-fixed paraffin embedded (FFPE) tissue blocks and processed according to the instructions of the manufacturer. The assay was run on the resultant lysates.Results:The overall percentageagreement between the Xpert® STRAT4 assay and IHC / FISH results were 85.15% for ESR, 89.90% for PGR,91.09% for ERBB2, 90.72% for MKI67 (when using a cut off of 10%) and 84.54% for MKI67 (when using a cut-off of 20%). The positive percentage agreement for ESR, PGR, ERBB2, MKI67 with 10% cut-off andMKI67 with 20% cut-offwere 82.76%, 94.64%, 68.97%, 91.30% and 96.05%, respectively, and the negative percentage agreement were 100%, 84.09%, 91.67%, 80.00% and 42.86%, respectively. Conclusion:The studyhas shownthattheXpert® BreastCancer STRAT4 assay shows good concordance with IHC and FISH in detecting breast cancer biomarkers, and may become a supplementary or alternativestandard of care aftervalidation studies are performed.
Development of a novel pre-screen algorithm for cardio-metabolic risk management using a genomics database resource
(Stellenbosch : Stellenbosch University, 2016-03) Luckhoff, Hilmar Klaus; Kotze, Maritha J.; Janse van Rensburg, Susan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Anatomical Pathology.
ENGLISH ABSTRACT: The timely assessment and treatment of dyslipidaemia is an important component of cardiovascular risk screening and intervention. The apolipoprotein E (APOE) ε-2/ε-3/ε-4 polymorphism associated with impaired lipid homeostasis provides a genetic link between cardiovascular disease (CVD) and late-onset Alzheimer’s disease (AD). Realization that the phenotypic expression of the risk associated APOEε-2 and ε-4 alleles may be dependent on non-genetic factors supports the inclusion of APOE genotyping in chronic disease screening programs. The lack of well-defined selection criteria for APOE genotyping, however, limits the use of this biomarker in clinical practice. The aim of the present study was to develop a pre-screen algorithm for identification of a target population most likely to benefit from APOE genotyping, performed in conjunction with a clinical and lifestyle assessment. Towards this goal, comprehensive patient data were evaluated from a total of 580 unrelated Caucasians enrolled in a chronic disease screening program over a five-year period (2010-2015), using an ethically approved study questionnaire. Biochemical tests performed according to standard laboratory protocols were extracted from the research database. All study participants were genotyped for the APOE ε- 2/ε-3/ε-4 polymorphism. APOE genotype distribution differed significantly (p<0.05) between study participants with and without a family history of AD. A positive association between dietary fat intake and lowdensity lipoprotein (LDL) cholesterol (p=0.001), as well as an inverse association with highdensity lipoprotein (HDL) cholesterol (p=0.002), were observed in patients with a family history of AD. Body mass index (BMI) was positively associated with LDL cholesterol and inversely associated with HDL cholesterol levels (p<0.001), irrespectively of an AD family history. Smoking was associated with higher triglycerides (p<0.001) and lower HDL cholesterol levels (p=0.004) in the total study group. Alcohol intake was positively associated with BMI (p=0.008) as well as triglyceride levels (p=0.021) in patients with a positive family history of AD. The clinical expression of a hypercholesterolaemic phenotype in APOE ε-4 allele carriers, as well as apparent mitigation by regular physical activity, were dependent on the interaction between a family history of AD and APOE genotype (p<0.001). APOE ε-2 carriers without an AD family history showed a significant increase in triglyceride levels (p=0.014). The modulating influence of APOE ε-4 on the relationship between alcohol intake and BMI as well as total cholesterol levels was also dependent on the presence or absence of AD family history (p<0.05). This study resulted in the addition of a family history of AD as a novel component to the prescreen algorithm developed for selection of at-risk individuals prior to APOE genotyping performed as part of a chronic disease screening program. The lifestyle questionnaire used in this study furthermore facilitated interpretation of the clinical relevance of variation detected in the APOE gene. This is important to prioritize the use of lipid-lowering medication towards patients with severe subtypes of dyslipidaemia such as familial hypercholesterolaemia (FH), which remains largely undiagnosed and untreated in the highrisk South African population. Incorporating the research findings into clinical practice would suggest that physical activity may be the most effective risk reduction strategy in carriers of the APOEε-4 allele, as supported by international studies.
Evaluation of high-throughput methodology for multi-gene screening in patients with Non-Alcoholic Fatty Liver Disease (NAFLD)
(Stellenbosch : Stellenbosch University, 2011-12) Fisher, Leslie Reginald; Kotze, Maritha J.; Kruger, F. C.; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology.
ENGLISH ABSTRACT: Non-Alcoholic Fatty Liver Disease (NAFLD) is the most prevalent chronic liver disease in Western countries and is considered the hepatic manifestation of the Metabolic Syndrome (MetS). Its heterogeneous nature ranges from hepatic steatosis through steatohepatitis to advanced fibrosis and cirrhosis where the ingestion of significant amounts of alcohol has been excluded. The disease profile of NAFLD and its necro-inflammatory subset Nonalcoholic Steatohepatitis (NASH) were described in the parent study, which provided a clinically well-characterised patient cohort for the present investigation. South African patients with NASH had significantly higher mean serum cholesterol and triglyceride levels than those with fatty liver only. The objective of this study was to implement a high-throughput real-time polymerase chain reaction (PCR) method in our laboratory to enable the assessment of cardiovascular genetic risk factors in NAFLD patients. The specific aims were to determine the clinical utility and perform analytical validation of each mutation included in the multi-gene cardiovascular disease (CVD) screening assay. The Pathology Supported Genetic Testing (PSGT) concept developed at our department provides a practical approach to personalized medicine. The CVD multi-gene screen analyses key metabolic pathways relating to atherogenic dyslipidaemia, chronic inflammation, hypercoagulation and iron dysregulation implicated in insulin resistance, which is known to be a universal factor in the pathogenesis of NAFLD. Deleterious low-penetrance mutations in the APOE (APOE2 and E4 alleles), MTHFR (677C>T and 1298A>C), F2 (20210G>A), FV (1691G>A, Leiden) and HFE (C282Y and H63D) genes were included for analysis due to their important role as genetic contributors to these biological processes. A total of 178 patients diagnosed with NAFLD and 75 controls were studied using direct DNA sequencing and a RT-PCR system for mutation detection. In addition, two patients with high ferritin levels were included as case studies. A significant association was found between HFE mutations and elevated Alanine Transaminase (ALT) levels in the NAFLD population (p = 0.04). This discovery is interpreted as the identification of a subset of patients at greater risk of developing progressive liver damage who would benefit most from genetic testing to direct more aggressive therapy at an earlier stage. The necessity of an integrative, systems-based network approach was demonstrated to more accurately distinguish between Hereditary Haemochromatosis (HH) and Insulin Resistance-associated Hepatic Iron Overload (IR-HIO) syndrome in obese patients. The PSGT approach to personalized medicine facilitates diagnosis of CVD subtypes, prevention of cumulative risk and the formulation of gene-based intervention programs tailored to the needs of the patient. These findings support the clinical utility of the CVD multi-gene test to guide chronic disease risk management in patients with NAFLD. The HFE mutation detection component of this test is of particular relevance in directing an effective treatment strategy in patients with a medical history of CVD and/or high iron stores.
Genetic aspects of pre-eclampsia : mutation screening of the low-density lipoprotein receptor, methylenetetrahydrofolate reductase, prothrombin and factor V candidate genes
(Stellenbosch : Stellenbosch University, 2001-03) Gebhardt, G. S.; Odendaal, H. J.; Hillermann, R.; Kotze, Maritha J.; Stellenbosch University. Faculty of Medicine & Health Sciences. Dept. of Pathology. Anatomical Pathology.
ENGLISH ABSTRACT: Pre-eclampsia is a condition unique to pregnancy and primarily affects the maternal and placental vascular endothelium. It has significant morbidity and mortality consequences for both mother and infant. Despite global research into the aetiology of the condition, the cause for this condition remains unknown. Several factors, including a strong family history of hypertension in pregnancy point to a familial or genetic component in the pathophysiology of this complication. The purpose of this research project was to investigate candidate genes implicated in endothelial damage. Common methylene-tetra-hydrofolate reductase (MTHFR) gene mutations C677T and A1298C, factor V Leiden mutation R506Q and prothrombin mutation A20210G were investigated in 50 patients with an uncomplicated pregnancy outcome (controls) and 350 patients with various clinical manifestations of preeclampsia, including severe, early onset forms and abruptio placentae. Fasting homocystein levels were determined biochemically on all participants. In addition, 126 consecutive pregnant patients were recruited at booking, fasting lipograms were performed on them as well as mutation screening of 7 common mutations in the low-density lipoprotein receptor gene. This was correlated with eventual pregnancy outcome, and those with an uncomplicated outcome were selected as an additional control group. A significant association between hyperhomocysteinaemia and early onset severe pre-eclampsia could be demonstrated. Mutant allele T of the C677T mutation could be associated with hyperhomocysteinaemia but not with pre-eclampsia whilst mutant allele C of mutation A1298C demonstrated a significant correlation with diastolic blood pressure. In addition, combined heterozygosity for these mutations may serve as a marker for abruptio placentae.
The MED-PED project : presymptomatic diagnosis in families with disease- related LDL receptor gene mutations
(Stellenbosch : Stellenbosch University, 2000-03) Vergotine, Joseph Vincent; Kotze, Maritha J.; De Jong, G.; Stellenbosch University. Faculty of Medicine & Health Sciences . Dept. of Pathology.
ENGLISH ABSTRACT: Familial hypercholesterolaemia (FH) contributes significantly to the high death rate from cardiovascular disease worldwide. FH is a common autosomal co-dominant disease characterised by raised cholesterol levels and premature coronary heart disease (CHD). Whilst these features usually are very prominent in homozygotes the clinical diagnosis of heterozygotes is complicated by variable phenotypic expression. Specific founder genes in the low-density lipoprotein receptor (LDLR) gene have increased the prevalence of FH in South African Afrikaners, Indians, Jews and Coloureds, and screening for these known mutations allows unequivocal diagnosis of FH-affected individuals. The systematic molecular analysis of FH resulted in the identification of at least ten founder-type LDLR gene mutations among the 56 different gene defects described to date in the diverse South African population. DNA screening of 792 at-risk family members for the FH-related mutations identified in 379 index cases, allowed accurate disease diagnosis in an additional 340 relatives and exclusion of the relevant mutation in 452 individuals. This effort forms part of the MED PED FH initiative, a collaborative project to "Make Early Diagnosis and Prevent Early Deaths in MEDical PEDigrees with FH". Evaluation of clinical criteria versus DNA diagnosis of three founder-related mutations (D154N, D206E and V408M) in the South African population demonstrated that the sensitivity and specificity of diagnoses, based on total cholesterol values measured in family members of index cases recruited for this study, were 88% and 77%, respectively. A population-directed DNA diagnosis of FH is therefore justified in South Africa on a routine basis, since expression of the defective gene measured in biochemical tests does not allow accurate diagnosis of FH in all cases. The application of mutation detection was illustrated by prenatal diagnosis of FH performed for a couple who are both heterozygous for the most common Afrikaner mutation, D206E. The mutation was absent in the foetus and a normocholesterolaemic infant was born. Prenatal diagnosis of FH, aimed at the detection of homozygous cases, is particularly applicable in populations and families with molecularly defined LDLR gene mutations. The MED-PED approach resulted in accurate diagnosis and subsequent treatment of FH in more patients, and referral to lipid clinics where they could receive the intensive care their condition justifies. Molecularly diagnosed FH patients will be the first to benefit from future treatment approaches based on mutation type.
Molecular-genetic analysis of Hirschsprung's disease in South Africa
(Stellenbosch : Stellenbosch University, 2000-03) Julies, Monique G.; Moore, S. W.; Kotze, Maritha J.; Du Plessis, L.; Stellenbosch University. Faculty of Medicine & Health Sciences. Dept. of Pathology.
ENGLISH ABSTRACT: Hirschsprung's disease, or aganglionic megacolon, is a common cause of intestinal obstruction in neonates and is associated with the congenital absence of intrinsic ganglion cells in the myenteric and submucosal plexuses of the gastrointestinal tract. The affected area is usually restricted to the distal part of the colon (short segment disease), but total colonic or intestinal involvement occurs in some patients (long segment disease). DNA analysis was performed on samples from 53 unrelated sporadic HSCR patients to search for mutations in RET proto-oncogene, endothelin-B receptor (EDNRB) and endothelin-3 (EDN3) genes. The patients were from different ethnic groups in South Africa, including 29 coloured, 14 white (Caucasian) and 9 black individuals. The origin of 1 patient was unknown. PCR HEX-SSCP analysis of the RET protooncogene revealed one previously described (P973L) and five novel mutations (V202M, E480K, IVS10-2A1G, D771N, IVS19-9Crr), likely to cause or contribute to the HSCR phenotype. Nine polymorphisms were also identified in the RET protooncogene, of which four were novel (IVS6+56deIG, IVS13-29Crr, IVS16-38deIG, X1159) and five previously described (A45, A432, L769, S904, R982). All the mobility shifts detected in the EDNRB gene represented polymorph isms (A60T, S184, 1187, V234, L277, IVS3-6Crr, IVS4+3A1G). No sequence variants were identified in the EDN3 gene. The majority of mutations in the RET proto-oncogene (28.6%) were identified in coloured patients while no mutations were identified in black patients. A mutation in RET was identified in two of 14 patients (14%) presenting with HSCR and Down's syndrome compared to 6 mutations identified in 9 of 39 patients (23%) with only HSCR. The fact that Down's syndrome patients have a high chance of developing HSCR, implies the involvement of modifier gene(s) in a HSCR/Oown's syndrome phenotype. This study demonstrated that, within the South African HSCR patient population, the RET proto-oncogene is the major susceptibility gene, whereas EDNRB and EDN3 may contribute only to a minority of cases. In 81% of patients no disease-causing mutation could be identified, which is in keeping with the heterogeneous nature of HSCR. The identification of mutations in HSCR patients would in future lead to improved and accurate counselling of South African HSCR patients and their families.

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Browsing Masters Degrees (Anatomical Pathology) by browse.metadata.advisor "Kotze, Maritha J."

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