Browsing by Author "Ramesar, Rajkumar"
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- ItemOpen AccessArrhythmogenic right ventricular cardiomyopathy type 6 (ARVC6): support for the locus assignment, narrowing of the critical region and mutation screening of three candidate genes(BioMed Central Ltd, 2006) Matolweni, Luzuko; Bardien, Soraya; Rebello, George; Oppon, Ekow; Munclinger, Miroslav; Ramesar, Rajkumar; Watkins, Hugh; Mayosi, BonganiBACKGROUND:Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a heritable disorder characterized by progressive degeneration of right ventricular myocardium, arrhythmias and an increased risk of sudden death at a young age. By linkage analysis, ARVC type 6 was previously mapped to a 10.6 cM region on chromosome 10p12-p14 in a large North American kindred. To date, the genetic defect that causes ARVC6 has not been identified. METHODS: We identified a South African family of 13 members with ARVC segregating as an autosomal dominant disorder. The diagnosis of ARVC was based on international diagnostic criteria. All available family members were genotyped with microsatellite markers at six known ARVC loci, and positional candidate gene screening was performed. RESULTS: Genetic linkage and haplotype analysis provided lod scores that are highly suggestive of linkage to the ARVC6 locus on chromosome 10p12-p14, and the narrowing of the critical region to ~2.9 Mb. Two positional candidate genes (ITG8 and FRMD4A) were screened in which defects could possibly disrupt cell-cell adhesion. A non-positional candidate gene with apoptosis inducing properties, LAMR1P6 (laminin receptor 1 pseudogene 6) was also screened. Direct sequencing of DNA from affected individuals failed to detect disease-causing mutations in the exonic sequences of the three genes investigated. CONCLUSION: The narrowing of the ARVC6 critical region may facilitate progress towards the identification of the gene that is involved in ARVC. Identification of the causative genes for ARVC will contribute to the understanding of the pathogenesis and management of this poorly understood condition.
- ItemOpen AccessCommon ABCA4 mutations in South Africans: frequencies, pathogenicity and genotype-phenotype correlations(2010) Nossek, C; Ramesar, Rajkumar; Greenberg, Jacquie
- ItemOpen AccessComputational analysis of candidate disease genes and variants for salt-sensitive hypertension in indigenous Southern Africans(Public Library of Science, 2010) Tiffin, Nicki; Meintjes, Ayton; Ramesar, Rajkumar; Bajic, Vladimir B.; Rayner, BrianMultiple factors underlie susceptibility to essential hypertension, including a significant genetic and ethnic component, and environmental effects. Blood pressure response of hypertensive individuals to salt is heterogeneous, but salt sensitivity appears more prevalent in people of indigenous African origin. The underlying genetics of salt-sensitive hypertension, however, are poorly understood. In this study, computational methods including text- and data-mining have been used to select and prioritize candidate aetiological genes for salt-sensitive hypertension. Additionally, we have compared allele frequencies and copy number variation for single nucleotide polymorphisms in candidate genes between indigenous Southern African and Caucasian populations, with the aim of identifying candidate genes with significant variability between the population groups: identifying genetic variability between population groups can exploit ethnic differences in disease prevalence to aid with prioritisation of good candidate genes. Our top-ranking candidate genes include parathyroid hormone precursor ( PTH ) and type-1angiotensin II receptor ( AGTR1 ). We propose that the candidate genes identified in this study warrant further investigation as potential aetiological genes for salt-sensitive hypertension.
- ItemOpen AccessDelineation of the genetic causes of complex epilepsies in South African pediatric patients(2023) Esterhuizen, Alina; Ramesar, Rajkumar; Wilmshurst JoanneBackground Sub-Saharan Africa bears the highest burden of epilepsy worldwide. A proportion is presumed to be genetic, but this aetiology is buried under the burden of infections and perinatal insults, in a setting of limited awareness and few options for testing. Children with developmental and epileptic encephalopathies (DEEs), are most severely affected by this diagnostic gap, as the rate of actionable findings is highest in DEE-associated genes. This research study investigated the genetic architecture of epilepsy in South African (SA) children clinically diagnosed with DEE, highlighting the clinical utility of informative genetic findings and relevance to precision medicine for DEEs in a resource-constrained setting. Methods A group of 234 genetically naïve SA children with drug-resistant epilepsy and a diagnosis or suspicion of DEE, were recruited between 2016 and 2019. All probands were genetically tested using a DEE gene panel of 71 genes. Of the panel-negative probands, 78 were tested with chromosomal microarray and 20 proband/parent trios underwent exome sequencing. Statistical comparison of electroclinical features in children with and without candidate variants was performed to identify characteristics most likely predictive of a positive genetic finding. Results Pathogenic/likely pathogenic (P/LP) variants were identified in 41/234(17.5%) * probands. Of these, 29/234(12.4%) * were sequence variants in epilepsy-associated genes and 12/234(5.1%) * were genomic copy number variants (CNVs). Sixteen variants of uncertain significance (VUS) were detected in 12 patients. Of the 41 children with P/LP variants, 26/234(11%) had variants supporting precision therapy. Multivariate regression modelling highlighted neonatal or infantile-onset seizures with movement abnormalities and attention difficulties as predictive of a positive genetic finding. This, coupled with an emphasis on precision medicine outcomes, was used to propose the pragmatic “Think-Genetics” decision tree for early recognition of a possible genetic aetiology, pragmatic testing, and multidisciplinary consultation. Conclusion The findings presented here emphasise the relevance of an early genetic diagnosis in DEEs and highlight the importance of access to genetic testing. The “Think-Genetics” strategy was designed for early recognition, appropriate interim management, and genetic testing for DEEs in resource constrained settings. The outcomes of this study emphasise the pressing need for augmentation of the local genetic laboratory services, to incorporate gene panels and exome sequencing. *These percentages were rounded off to whole numbers in the published articles included in this thesis (i.e., rounded off to 18%, 12% and 5%, respectively).
- ItemOpen AccessElucidating the genetic aetiology of Bipolar Disorder(2018) Engelbrecht, Hannah-Ruth; Ramesar, Rajkumar; Dalvie, ShareefaIntroduction: Bipolar disorder (BD) is a debilitating mood disorder with substantial genetic contributions. However, while the existence of its heritability is well-established, the precise genetic components and mechanisms of BD remain unknown. This is a pilot study aimed at optimising the use of small-scale next generation sequencing (NGS) of family members affected with BD and extending the finding of variants to larger scale association studies, and an attempt at replicating associations from a large genome-wide association study. Methods and Materials: This thesis describes the pathway analysis of whole-genome sequencing (WGS) data from four Afrikaner individuals to identify candidate variants for genotyping in a Family-Based Association Test (FBAT) of 621 individuals of Caucasian and Mixed Ancestry families from South Africa. This was followed by whole-exome sequencing (WES) of eight members of an Afrikaner family to identify rare, coding variation. These two approaches were used to identify both common and rare variation which may be involved in BD. Results: FBAT indicated that variants in the genes ACTN2 (rs4659702) and ANK3 (rs10994318) are associated with BD in a combined group of both Mixed Ancestry and Caucasian individuals, p = 0.0339 and 0.0443, respectively. Furthermore, this study identified a variant in ACTN2 (rs11355106) which was associated with a broad psychiatric phenotype in Mixed Ancestry families (p = 0.0083). WES revealed 168 exomic variants that were shared by five affected members of the family, one of which was rs142375896, a rare and potentially damaging missense variant in SLC26A9. Conclusions: Pathway analysis of both WGS and WES data implicated that the burden of variation in affected individuals lies in regulatory networks, including the regulation of the actin cytoskeleton and circadian entrainment pathways. The association of ANK3 (rs10994318) with BD in a European ancestry cohort was replicated in a South African cohort comprising of Caucasian and Mixed Ancestry individuals, indicating that some risk variants for the disorder could be shared across populations. This thesis confirms the validity of relatively small-scale family-based studies for the study of complex disorders.
- ItemOpen AccessEpidemiology and genetic risk factors of suicidal behaviour in South Africa(2023) Kootbodien, Tahira; Ramesar, Rajkumar; Martin LornaBackground: Suicide is an urgent public health problem. Fatal suicidal behaviour (individuals who died by suicide) and non-fatal suicidal behaviour (attempted suicide, self-harm, and suicidal ideation) comprise a complex interplay of individual, social, environmental, and biological factors, that are not fully understood. Given the considerable societal cost associated with suicide and health inequality in South Africa, there is a critical need to determine the burden of suicide and risk factors associated with suicide, to understand who is most at risk to inform effective prevention efforts. Despite clear evidence of multiple risk factors, suicidal behaviour remains difficult to predict and prevent. Prevention efforts in South Africa may be limited by the lack of a national suicide prevention plan and the low base rate of individuals who died by suicide may impede research comparing fatal and non-fatal suicidal behaviour in settings such as ours. In addition, existing suicide data are derived primarily from high-income countries rather than lowand middle-income countries (LMICs) where suicide and poverty levels are high, and the mental health treatment gap is large. The purpose of this study was to broaden our understanding of suicidal behaviour in South Africa by combining various data sources, each representing a unique perspective of the problem, to build on existing knowledge that may inform suicide prevention strategies in South Africa. This thesis is organised into four studies and aimed to investigate risk factors associated with suicidal behaviour and to identify opportunities for targeted suicide prevention. The specific objectives of each study component were as follows: • To describe trends and demographic risk factors in deaths from suicide as well as other conditions that may include suicide to identify populations at risk in South Africa (Study 1). • To investigate the association between environmental and occupational organophosphate pesticide (OP) exposure and attempted suicide in adults admitted to hospital in Cape Town, South Africa (Study 2). • To explore the genetic architecture underlying suicidal behaviour and psychiatric disorders to understand the genetic factors that increase the risk of suicidal behaviour (Study 3). • To describe healthcare utilisation 12 months before suicidal behaviour among individuals who attempted suicide and who died by suicide, to identify opportunities for prevention in Cape Town, South Africa (Study 4). Methods: This thesis included an ecological time-series study of national suicide mortality data from Statistics South Africa using joinpoint regression analysis (Study 1, N=10.3 million recorded deaths from 1997 to 2016; 8,573 deaths from suicide); a conditional logistic regression analysis of an attempted suicide hospital-based case-control study (Study 2, N=400; 200 cases and controls); a genome-wide genetic correlation study of suicidal ideation, self-harm, attempted and fatal suicide (samples [n] ranged from 62,648 to 125,844), and selected psychiatric disorders (n ranged from 9,954 to 386,533) using a genomic structural equation modelling approach (Study 3); and a retrospective cohort of linked electronic health records of individuals who attempted suicide and were admitted to hospital and a case series of fatal suicides on whom forensic autopsies were performed at a mortuary in Cape Town (Study 4, N=484). Results: The key findings in this study show that (i) suicide mortality rates were consistently higher in men than women between 1997 and 2016 (Study 1). Suicide rates increased by 7.7% among young people aged 15 to 29 years. Hanging, poisoning and firearms were the most frequent methods of suicide used. Subgroup analysis showed suicide by hanging and poisoning mortality rates increased by 2.9% and 3.7% across 20 years. Suicide deaths were underreported and may be included among deaths by accidental injuries and undetermined intent. However, these patterns varied by method of death (hanging, poisoning and firearm injury) over the study period. The largest proportion of suicide deaths may be potentially misclassified as accidental hanging and hanging by undetermined intent, and to a lesser extent, accidental poisoning and poisoning by undetermined intent. In contrast, firearm-related deaths were more likely to indicate a homicide than a suicide death. Missing data on select sociodemographic variables limited the accuracy and generalisability of our findings. (ii) Pesticide use in homes and gardens was common (85%); however, there was no association between attempted suicide and environmental (household, garden, and occupational) OP exposure (Study 2). Hazardous drinking and unemployment with no household income were significantly associated with an increased risk of attempted suicide while sharing the house with more than three persons was protective. (iii) We observed strong significant genetic correlations (rg) between suicidal ideation, attempted suicide, and self-harm (rg range, 0.71 to 1.09) and moderate-to-strong genetic correlations between suicidal behaviour traits and a range of psychiatric disorders (Study 3). The strongest genetic correlation was noted for major depressive disorder and suicidal ideation (Ever contemplated self-harm, rg=0.86±0.07, p=1.62x10-36). Multivariate genomic analysis revealed a single (common) factor structure for suicidal behaviour traits, major depressive disorder, attention-deficit hyperactivity disorder (ADHD), and alcohol use disorder. Approximately 2,951 genes and 98 sub-network hub genes were associated with the common factor, and shared biological pathways include involvement in developmental biology, signal transduction and RNA degradation. (iv) Approximately two-thirds of cases had at least one prior visit to a health care facility in the 12 months leading to suicidal behaviour (Study 4). The prevalence of psychiatric disorders was lower for individuals who died by suicide than attempted suicides but both groups interacted equally (approximately 65%) with the healthcare system during the 12 months leading to suicidal behaviour. Patients who used primary care services in the year before dying by suicide attended for the management of their chronic conditions (such as cardiovascular disease and diabetes) and emergency medical care for assault-related injuries. For attempted suicides, common reasons for a healthcare visit were for management of their chronic condition, HIV care, and a psychiatric diagnosis of depression, bipolar, or substance use disorders. Conclusions: This study expands on previous research and shows that while common risk factors are shared by individuals with fatal and non-fatal suicidal behaviour, the degree of risk varies across suicide groups, age, and sex. Findings of increased genetic risk of suicidal behaviour among individuals with psychiatric disorders suggest that identification and early treatment of co-morbid psychiatric disorders (major depression, alcohol use disorder, ADHD, and schizophrenia) should be included in suicide prevention strategies. Evidence of potential misclassification of suicide death within accidental injuries and undetermined intent categories may explain the underestimation of suicide mortality reported in this study. Combined with the high proportion of missing data in the national vital statistics and poor data quality of external causes of death, these findings suggest a critical need for ongoing training on the cause of death certification and further interventions to improve suicide data quality. Further, continued monitoring of suicide mortality data and linking electronic health records may provide opportunities for suicide surveillance that can help identify where prevention strategies should be allocated for maximum benefit, such as primary healthcare outpatient facilities, emergency treatment centres, and antiretroviral clinics.
- ItemOpen AccessGenetic analysis of bipolar disorder and alcohol use disorder(2015) Dalvie, Shareefa; Ramesar, Rajkumar; Stein, Dan JBackground: Mental health disorders represent a major public health problem in most countries around the world. In South Africa, the lifetime prevalence of psychiatric disorders is 30.3%, with substance-use disorders and mood disorders being the second and third most prevalent classes of lifetime disorders, respectively. Bipolar disorder (BD) has a lifetime prevalence of 1.4% and alcohol use disorder (AUD) a lifetime prevalence of 30.3%, and they are frequently comorbid. Both of these disorders have a relatively high heritability, yet the exact genetic basis of each remains unknown. Genetic variants within the hypothalamic-pituitary-adrenal (HPA)-axis and glutamatergic pathways have previously been implicated in both phenotypes. The aim of this project was to investigate the aetiology of BD and AUD, using high-throughput genomic technologies, bioinformatics, brain-imaging and environmental measures. An additional aim was to assess the genetic aetiology of BD-AUD comorbidity. Methods: For the genetic analysis underlying BD, a South African 'Afrikaner' family was investigated. Whole-genome sequencing (WGS) and whole-genome linkage analysis was performed for individuals with BD Type I (BDI) and unaffected family members using the Illumina HiSeq2000 and Affymetrix Axiom TM Genome-wide CEU 1 Array, respectively. For the AUD analysis, two groups were investigated; a South African adolescent group comprising 80 individuals with AUD and 80 controls, and a group of 8123 individuals from the Avon Longitudinal Study of Parents and Children (ALSPAC) birth cohort. The South African group of adolescents were genotyped using the Illumina Infinium iSelect custom 6000 BeadChip, childhood trauma data was obtained and brain magnetic resonance images were collected for a subset of this group. Genotype data on HPA-axis genes were obtained from a previous study for the ALSPAC cohort. The fourth group of individuals investigated in this thesis comprised 233 individuals with BD-AUD comorbidity from the Systemic Treatment Enhancement Program for BD (STEP-BD). Genotype data for genes from the glutamatergic and HPA-axis pathways were obtained from a previous study conducted on these individuals. Results: The chromosomal regions 6p25, 10p14-10p15.1, 11q23-11q25, and 13q21-22 scored the highest LOD scores for BD and the most over-represented pathway in the affected family members was the T-cell receptor signalling pathway. In the South African adolescent group, circadian rhythm genes were associated with AUD and childhood trauma predicted alcohol use in adolescence. The gene-imaging analysis identified a SNP in the glutamate receptor, ionotropic, N-methyl D-aspartate 2B (GRIN2B) gene as being associated with brain volume in the left orbitofrontal cortex and posterior cingulate. HPA-axis genes did not show an association with AUD and no significant gene x environment interactions were detected for AUD in the ALSPAC cohort. Single variants in the glutamatergic genes and HPA-axis were not associated with BD-AUD comorbidity. However, from the gene-based analysis, the glutamatergic gene PRKCI was associated with BD-AUD comorbidity. Conclusions: It appears that disruption in immune-related genes may contribute to the development of BD in an Afrikaner family. No significant gene x environment interactions were detected for adolescent AUD. The circadian pathway and childhood trauma may play a role in the development of adolescent AUD. Differential brain volume and BD-AUD comorbidity may be characterised by variation in the glutamatergic pathway. These pathways and the interactions between them should be further investigated in BD and AUD.
- ItemOpen AccessGenetic analysis of inherited retinal diseases in indigenous Southern African populations(2017) Roberts, Lisa Jane; Ramesar, Rajkumar; Swaroop, AnandBackground: Inherited retinal diseases (IRDs) constitute a group of clinically and genetically heterogeneous conditions which cause degeneration of retinal photoreceptor cells and result in visual impairment. Characterisation of the genetic basis of IRD is not only beneficial for the affected families, but also contributes towards understanding of the disease pathobiology. Investigations into the molecular basis of IRDs have been ongoing in South Africa (SA) for over 30 years, however the evaluation of reported genetic mutations has yielded low returns in certain populations. Indigenous southern Africans comprise a unique population group with distinct genetic diversity, providing a valuable resource for genetic discoveries; nonetheless, this population remains largely underrepresented in genomic studies. The aim of this investigation was to characterise the underlying genetic mutations in a cohort of indigenous African IRD patients. Methods: The IRD registry in the Division of Human Genetics (University of Cape Town) was reviewed for causative mutations. Subsequently, upon identifying a mutation underlying Usher Syndrome in two indigenous African patients, an assay was designed to screen for this mutation in probands with different IRDs (n=170) and controls (n=51), and haplotype analysis was performed on mutation-positive individuals. The registry review additionally served to identify a suitable cohort for the application of next generation sequencing (NGS) technology. Whole exome sequencing (WES) was performed on genomic DNA samples from 56 individuals from 16 families. The WES data analysis strategy involved prioritisation of variants in reported and candidate IRD genes. Rare, co-segregating, pathogenic, exonic or splice variants were validated by Sanger sequencing. Custom TaqMan assays were designed to screen seven mutations, identified by WES, in 193 unrelated indigenous African probands with IRDs. Results: A homozygous founder mutation, c.6377delC in MYO7A, was identified in 43% of the indigenous African patients with Usher syndrome, which is the most common cause of deaf-blindness. Targeted WES data analysis of all known IRD genes resulted in identification of the underlying genetic defects in six distinct genes (RHO, PRPF3, PRPF31, ABCA4, CERKL, and PDE6B) in six families. Taqman screening revealed four additional probands with identical homozygous mutations in CERKL and PDE6B. An X-linked gene (RP2) mutation was subsequently identified in an affected family with semi-dominant retinitis pigmentosa. Supplementary analysis of the X-linked RPGR ORF15 mutation hotspot (not adequately covered by WES) identified two mutations in three families. A novel IRD gene, IDH3A, was found in one family by analysis of 22 putative candidate genes. The large number of variants in the remainder of the indigenous African exomes presented considerable challenges for identification of additional novel genes. Discussion: The results of this project have important implications for IRD molecular diagnostic services in SA. Using WES, a genetic diagnosis was obtained for ±73% of the indigenous African cohort, and ±70% of the causative mutations identified were novel. This outcome emphasises the superiority of NGS-based approaches over genotyping-based microarrays which screen for IRD mutations previously reported in other (mainly European-derived) populations. The unexpected identification of mutations in known X-linked genes in four families highlighted key considerations for IRD WES analysis. Cascade screening of mutations identified in this study, across larger cohorts of unrelated probands, revealed the genetic cause of IRD in additional cases and the number of indigenous African families in the registry with a genetic diagnosis was effectively doubled. Members of these families can now opt for diagnostic, carrier, or predictive testing of familial mutations. Finally, the information obtained from this research contributes towards a better understanding of the genetic architecture of IRDs in SA.
- ItemOpen AccessGenetic variation at selected SNPs in the leptin gene and association of alleles with markers of kidney disease in a Xhosa population of South Africa(Public Library of Science, 2010) Okpechi, Ikechi G; Rayner, Brian L; van der Merwe, Lize; Mayosi, Bongani M; Adeyemo, Adebowale; Tiffin, Nicki; Ramesar, RajkumarBACKGROUND: Chronic kidney disease (CKD) is a significant public health problem that leads to end-stage renal disease (ESRD) with as many as 2 million people predicted to need therapy worldwide by 2010. Obesity is a risk factor for CKD and leptin, the obesity hormone, correlates with body fat mass and markers of renal function. A number of clinical and experimental studies have suggested a link between serum leptin and kidney disease. We hypothesised that variants in the leptin gene ( LEP ) may be associated with markers of CKD in indigenous black Africans. METHODOLOGY/PRINCIPAL FINDINGS: Black South Africans of Xhosa (distinct cultural Bantu-speaking population) descent were recruited for the study and four common polymorphisms of the LEP (rs7799039, rs791620, rs2167270 and STS-U43653 [ENSSNP5824596]) were analysed for genotype and haplotype association with urine albumin-to-creatinine ratio (UACR), estimated glomerular filtration rate (eGFR), Serum creatinine (Scr) and serum leptin level. In one of the four single nucleotide polymorphisms (SNPs) we examined, an association with the renal phenotypes was observed. Hypertensive subjects with the T allele (CT genotype) of the ENSSNP5824596 SNP had a significantly higher eGFR (p = 0.0141), and significantly lower Scr (p = 0.0137). This was confirmed by haplotype analysis. Also, the haplotype GAAC had a modest effect on urine albumin-to-creatinine ratio in normotensive subjects (p = 0.0482). Conclusions/Significance These results suggest that genetic variations of the LEP may be associated with phenotypes that are markers of CKD in black Africans.
- ItemOpen AccessGenetics of age-related macular degeneration and Stargardt disease in South African populations(2016) Baard, Johannes; Ramesar, Rajkumar; Roberts, LisaBackground: The Retinal Degenerative Diseases (RDD) Research Group in the Division of Human Genetics at UCT has for the past 25 years been intensively investigating a range of RDD phenotypes. Two points of particular note have emerged regarding Macular Degenerations (MD) : (i) that more than 58% of juvenile MD, notably Stargardt Disease (STGD) , in Caucasian populations may have the underlying causative genetic defect identified , while only 1 1 % of the similar phenotype in indigenous African populations is resolved, and (ii) that the 'elderly' form of MD, i.e. age - related macular degeneration (AMD) has a remarkably lower incidence in the indigenous African population when compared to any other population group, and most notably the Caucasian (or European - derived) population /s . This study investigates the genetic factors underlying macular degeneration (MD) in our study cohort comprising various South African ethnolinguistic groups with particular focus on disease in juvenile and elderly indigenous Africans. Materials and Methods: For the STGD part of the study, sequencing of the entire ABCA4 coding and splice region (comprising 50 amplicons) was performed in three African STGD patients who were representative of three common haplotypes identified within the larger cohort of 36 patients . Pathogenicity predictive software, PON - P and Human Splice Finder (HSF), were used for in silico data analysis. For the AMD subset: Available local indigenous southern African population - based genome - wide S ingle Nucleotide Polymorphism (SNP) chip (Affymetrix SNP6) data was used to identify SNPs within known AMD candidate genes in which allele frequencies were significantly different (i.e. 10 fold) between Caucasians and indigenous southern Africans. Nine SNPs occurring at higher frequencies within Africans compared to Caucasian controls were genotyped by SNaPshot PCR within a multi - ethnic AMD SA cohort. Minor allele frequencies (MAF) were compared using SHEsis. Results: Sequencing of ABCA 4 in three African STGD patients produced 39 unique variants, out of which only one, (V643M), was deemed pathogenic. HSF predicted 22 of these non - exonic variants to be 'possibly pathogenic', confounding analysis. No variants segregated with the common haplotypes. Regarding the AMD cohort, eight SNPs in candidate AMD genes showed a decreased MAF in African AMD cases compared to controls, two of which (rs9621622 in TIMP3 and rs17110714 in ABCA4 ), were statistically significant ( p values of 9.95 x 10 - 4 and 1.04 x 10 - 2 , respectively). Discussion and Conclusion: Although a number of variants were identified in the coding region of three haplotype - representative STGD subjects, only one variant proved pathogenic but did not co - segregate with the haplotype in the rest of the samples. It is possible that variants in regulatory regions not captured by the exonic screening might be involved, or that another gene may be imp licated in the 'STGD - like' phenotype in the indigenous African subjects. In the second part of the study, the investigation of the African AMD cohort suggested that SNPs in TIMP3 and ABCA4 are associated with a decreased susceptibility, and may therefore plausibly be protective for AMD in indigenous Africans. Overall, however, this should be considered only a pilot study of macular degeneration in the indigenous African population, providing leads to larger scale studies of this group of disorders in this population group.
- ItemOpen AccessThe genetics of anthracycline-induced cardiotoxicity in cancer patients(2018) Naidoo, Horacia; Ramesar, Rajkumar; Simonds, Hannah MINTRODUCTION: Breast cancer makes up 25% of all cancers diagnosed worldwide. Despite an increasing yearly incidence, there has been a significant decrease in mortality owing to early diagnosis and advances in treatment. Anthracycline-based chemotherapy is a relatively low cost yet highly effective anti-cancer treatment, increasing survival from 30% to >80%, presently. However, treatment efficacy is marred by the increased risk of anthracycline-induced cardiotoxicity (ACT) - estimated at 10-26%. Internationally, there has been evidence of ACT having a genetic basis. Currently in South Africa, there is little information on ACT in cancer patients and survivors, and no information on the genetic basis of this phenomenon. Our recruitment sites in Cape Town - Groote Schuur Hospital (GSH) and Tygerberg Hospital (TBH), routinely treat hundreds of patients, notably with breast cancer, with anthracycline-based therapy every year, and provided the environment to assess ACT, as well as genetic factors which may influence this adverse drug reaction. Left ventricular ejection fraction (LVEF) acts as a surrogate measure of cardiac function in the public health-care setting. OBJECTIVES: To provide insight into the clinical management of breast cancer patients on anthracycline-based treatment with a focus on the prevalence of ACT. To provide an index of genetic susceptibility to ACT and potentially contribute to a personalized medicine approach for a genetically diverse population. METHODOLOGY: In the retrospective part of the study, the clinical records of cancer patients treated with anthracyclines from 2011- 2016 at the Oncology Clinic at GSH were analysed. Clinical co-morbidities such as hypertension, diabetes, pre-existing cardiac disease and smoking as well as type and dose of anthracyclines, cardiac function and patient status were assessed. In the prospective study, breast cancer patients treated with anthracyclines, with a pre and post-treatment LVEF measure were recruited at GSH and TBH from 2013 to 2016. Patients were consented for access to both clinical information and biological material. Demographics, clinical risk factors and chemotherapeutic regimen data were analysed. LVEF, biomarkers and clinical status were also assessed in terms of reflecting ACT. In some instances certain clinical information was not available (i.e. LVEF) and out of necessity, a statistical correlation model or classifier was created in order to use available clinical data to derive missing clinical measures. Patients' DNA were analysed for seven genetic variants in the following six genes ABCC1 (rs246221); ABCC2 (rs17222723; rs8187710); HNMT (rs17583889); NCF4 (rs1883112): RAC2 (rs13058338) and RARG (rs2229774), and tested for correlation with clinical status and cardiac injury. Finally, a corollary study was conducted on a subset of patients in an attempt to determine whether cardiac biomarkers may be more sensitive measures of cardiotoxicity. RESULTS & DISCUSSION: In the retrospective cohort (n=402) 19.7% of patients showed diminished cardiac function. Logistic regression showed that the following predictors: type of first line chemotherapy, and total dose significantly contributed to the ACT phenotype as measured by change in LVEF. In the prospective patients (n=272), 14% were affected with ACT, with an increased likelihood of cardiotoxicity in the Indigenous African population. Logistic regression showed that both total anthracycline dose and change in LVEF were predictive of ACT. In the association study of prospective patients, only the RARG rs2229774 variant was significantly associated with patient ACT status (p=0.049, Chi-Square Test). Forty-two patients were assessed for the β-Natriuretic Peptide (BNP) biomarker and showed limited utility in correlating clinical status and/or LVEF decrease in all patients except Indigenous Africans indicating potential increased susceptibility of population group to ACT. LVEF was found to be unreliable as significant LVEF decreases did not always correlate with cardiac impairment and vice-versa. Changes in routine clinical patient management and overburdening of the nuclear medicine department also translated to only one LVEF measure being obtained in some instances. The statistically derived classifier for missing indicators of heart function was useful, but will require refinement. CONCLUSIONS AND RECOMMENDATIONS: Despite the inability of genotype as a predictor of ACT in this study, the increased susceptibility in the Indigenous African population to ACT as well as increased BNP levels after chemotherapy requires a closer look. The interrogation of lndigenous African patient genomes for novel variants of susceptibility to ACT are recommended; this requires building up of a substantial cohort from this population group, which would likely require collaboration with health care institutions in one of the other provinces of South Africa e.g. Eastern Cape, KwaZulu-Natal and/or Gauteng. Both this study and literature recommend the need for clinical trials for new and existing drugs on local African populations for both safety and efficacy. Furthermore, the BNP biomarker may be better suited to the prediction of irreversible cardiac damage rather than early cardiotoxicity. Troponin, released in response to cardiomyocyte death, may be a more sensitive biomarker in predicting ACT. Similarly, the inherent variability and lack of sensitivity of LVEF as a measure of cardiac function warrants the consideration of alternatives such as echocardiography or tissue-doppler imaging. Findings derived from this study indicate the need for refined patient management of ACT in a South African population to potentially allow for treatment with minimised risk and event-free breast cancer survival.
- ItemOpen AccessA Genomic Portrait of Haplotype Diversity and Signatures of Selection in Indigenous Southern African Populations(Public Library of Science, 2015) Chimusa, Emile R; Meintjies, Ayton; Tchanga, Milaine; Mulder, Nicola; Seoighe, Cathal; Soodyall, Himla; Ramesar, RajkumarWe report a study of genome-wide, dense SNP (∼900K) and copy number polymorphism data of indigenous southern Africans. We demonstrate the genetic contribution to southern and eastern African populations, which involved admixture between indigenous San, Niger-Congo-speaking and populations of Eurasian ancestry. This finding illustrates the need to account for stratification in genome-wide association studies, and that admixture mapping would likely be a successful approach in these populations. We developed a strategy to detect the signature of selection prior to and following putative admixture events. Several genomic regions show an unusual excess of Niger-Kordofanian, and unusual deficiency of both San and Eurasian ancestry, which were considered the footprints of selection after population admixture. Several SNPs with strong allele frequency differences were observed predominantly between the admixed indigenous southern African populations, and their ancestral Eurasian populations. Interestingly, many candidate genes, which were identified within the genomic regions showing signals for selection, were associated with southern African-specific high-risk, mostly communicable diseases, such as malaria, influenza, tuberculosis, and human immunodeficiency virus/AIDs. This observation suggests a potentially important role that these genes might have played in adapting to the environment. Additionally, our analyses of haplotype structure, linkage disequilibrium, recombination, copy number variation and genome-wide admixture highlight, and support the unique position of San relative to both African and non-African populations. This study contributes to a better understanding of population ancestry and selection in south-eastern African populations; and the data and results obtained will support research into the genetic contributions to infectious as well as non-communicable diseases in the region.
- ItemOpen AccessGenomics of Lynch syndrome and Constitutional mismatch repair deficiency syndrome(2018) Lamola, Lindiwe; Ramesar, RajkumarIntroduction: The mismatch repair system plays an important role in maintaining the genome integrity as it functions to correct mismatches during DNA replication. Heterozygous mutations in one of the mismatch repair (MMR) genes e.g. MLH1, MSH2, MSH6 and PMS2 cause the dominant adult cancer syndrome termed Lynch syndrome (or hereditary non-polyposis colorectal cancer). In our South African cohort, the MLH1 exon 13 c.1528C>T mutation is the most common Lynch syndrome-causing variant in the Mixed Ancestry population. Recently, a patient homozygous for this mutation, diagnosed with Constitutional mismatch repair deficiency (CMMR-D) syndrome was described within this extended cohort. CMMRD syndrome results in an increased predisposition to a range of cancers, most commonly brain and hematological tumours in early childhood. The aims of this thesis were: (i) to determine the rate of extra-colonic cancers in the cohort of Lynch syndrome families in our colorectal cancer registry, (ii) to determine if MLH1 c.1528C>T is a founder mutation, and (iii) to focus on the CMMR-D syndrome as a branch of Lynch syndrome and to potentially use the hypermutability-status in CMMR-D to understand the diverse carcinogenesis in Lynch syndrome. Methods: The registry consisting of Lynch syndrome families was interrogated and analysed to address the aim (i). Haplotype analysis was performed using microsatellite markers around the MHL1 c.1528C>T mutation to determine founder effect for aim (ii). For aim (iii) whole exome sequencing was also performed in a Lynch/ CMMR-D syndrome family in order to investigate the extent of hypermutability in CMMR-D syndrome, and to develop a working hypothesis for carcinogenesis in CMMR-D and Lynch syndromes. Results: From the analysis of the registry it was noted that 396 individuals carried a disease-causing mutation in either MLH1 or MSH2; females have a relatively later age of onset (for cancer) than males and MLH1 mutation carriers develop cancers relatively earlier in life than in individuals with MSH2 mutations. The most common extra-colonic cancers were endometrial and breast in females; in males small bowel cancer was most common, after CRC. The cohort study revealed a large founder effect with the MLH1 c.1528C>T mutation, with the most common inferred (disease-associated) haplotype found in 25 of the 30 subjects tested; the disease-associated haplotype was not present in controls. The mutation aging analysis traced the mutation to be ~225 years old. The WES investigation of the nuclear family within which the CMMR-D patient, including acquired and germline mutations in tissues from the child with CMMR-D, revealed a range of pathways including the extracellular matrix, WNT signaling, TGFβ and p53 as acquiring significant numbers of variants as a result of the MMR deficiency. Discussion and Conclusion: The results which are indicative of the need to improve the Lynch syndrome mutation testing and management for all patients, also suggests the need to develop surveillance programs for extra-colonic cancers, which will improve compliance and disease-free survival. WES investigation of the nuclear family containing a child with CMMR-D point to the potential involvement of a range of pathways associated with cancer development which may be indirectly invoked in the process of tumorigenesis by the wide range of variants acquired as a result of mismatch repair deficiency. It is likely that some of these processes are also involved in the emergence of extracolonic cancers in individuals affected with Lynch syndrome (i.e. heterozygous for mutations in MMR genes).
- ItemOpen AccessInvestigating the genetic basis of cisplatin-induced ototoxicity in adult South African patients(2016) Spracklen, Timothy Francis; Ramesar, Rajkumar; Vorster, Anna AlveraCisplatin, a potent chemotherapeutic agent, is widely used in the treatment of numerous soft-tissue cancers. Although high cure rates can be achieved when cisplatin is incorporated in chemotherapy regimens, the therapeutic utility of the drug may be limited by the development of dose-limiting adverse reactions in patients. A prevalent reaction to cisplatin is ototoxicity, or drug-induced hearing loss, which occurs when the drug accumulates in and damages cells of the inner ear, leading to permanent and progressive hearing impairment. In this investigation, two approaches were employed to explore the role of genetics in cisplatin response amongst South African cancer patients (n = 214). Using a candidate gene approach, which investigated variants in six genes which are involved in drug transport and processing, potential modifiers in the genes nuclear factor, erythroid 2-like 2 (NFE2L2) and solute carrier family 22, member 2 (SLC22A2) were identified. SLC22A2 encodes a known transporter of cisplatin, and the variant rs316019 conferred potentially protective effects against Chang- and TUNE-graded ototoxicity through a reduced transport of the drug (p = 0.039 and p = 0.031, respectively). Similarly, the variant NFE2L2 rs6721961 was possibly protective, as it occurred more frequently in patients who did not develop hearing impairment according to four different ototoxicity grading scales during high-dose (≥ 200 mg/m2) cisplatin treatment (ASHA, p = 0.001; Chang, p = 0.022; CTCAE, p = 0.001; TUNE, p = 0.028). When supplementing the prospective cohort with retrospective patient data, an increased susceptibility of indigenous African patients to Chang grade > 0 ototoxicity was observed (p = 0.001). For this reason, whole-exome sequencing was conducted on a subset of the patient cohort (n = 11), focussing on individuals of African origin who represented the phenotype extremes. Potential genetic modifiers were identified in genes involved in various biological processes, including transmembrane transport, development, hearing, the response to DNA damage, immune reactions and signalling pathways, implicating many previously unreported genes in the cellular response to cisplatin as well as its ototoxicity. The results reported in this study indicate that genetic information can improve predictive models of cisplatin response, although there are many novel genes which should be explored in the South African population. Identifying these genetic modifiers, such as those in SLC22A2 and NFE2L2, has the potential to further our understanding of this adverse drug reaction, and may assist in the future personalisation of treatment plans in the management of cancer.
- ItemOpen AccessAn investigation into factors which have an impact on access to and utilisation of the genetic and endoscopic surveillance clinic offered to high-risk members of known Lynch families(2011) Bruwer, Zandré; Ramesar, RajkumarThe Genetic and Endoscopic Surveillance Clinic provides predictive testing and life-saving colorectal cancer screening services to individuals with Lynch syndrome in the Western and Northern Cape provinces of South Africa. The risk of colorectal cancer is reduced by 50% and mortality is decreased by 65% with regular colonoscopic screening; however, the attendance rate at the clinic has been declining over several years. Concerns exist for those individuals undergoing screening at levels below the desired recommendations. It was thus opportune for a formal evaluation of both the surveillance and predictive testing programmes to be conducted to determine factors affecting the access, utilisation and satisfaction with the service, from the perspective of the service users.
- ItemOpen AccessAn investigation into the molecular basis of familial forms of osteoarthropathy in South Africa(1998) Ballo, Robea; Beighton, Peter; Ramesar, RajkumarGeneralised osteoarthritis (OA) is a common disorder of the joints which can lead to pain and disability. Identification of the determinant gene(s) is limited in part by the lack of Mendelian inheritance in most forms of the disorder, the combination of genetic and environmental influences and the late development of the condition. An approach to the investigation of the aetiology of OA would be to take advantage of the monogenic basis of inherited skeletal dysplasias in which OA is a major component. For this reason, the molecular genetic basis of the epiphyseal dysplasias, which encompass a spectrum of phenotypes ranging from mild to severe skeletal involvement, is addressed in this thesis. Familial skeletal disorders in South Africa in which OA is a major feature were identified and investigated using intragenic and closely linked microsatellite markers in order to determine linkage to candidate genes. Mutational analysis was undertaken to identify the genetic defect.
- ItemOpen AccessThe molecular genetic and epidemiological investigation of colorectal cancer in South Africa(2003) Felix, Rebecca; Ramesar, Rajkumar; Bodmer, WalterCancer of the colon and rectum is the third-most common cause of death due to neoplasia in Western countries. Colorectal cancer (CRC) can broadly be divided into the hereditary, non-hereditary and sporadic forms. Hereditary cancers account for approximately 10% of the CRCs. Familial cancers can be divided into thos types in which polyps are a dominant feature (e.g. familal adenomatous polyposis-FAP) and the non-polypotic forms (e.g. hereditary nonpolyposis colorectal cancers-HNPCC).
- ItemOpen AccessA molecular investigation of the novel gene underlying autosomal dominant retinitis pigmentosa in a South African family(1999) Bardien-Kruger, Soraya; Greenberg, Jacquie; Beighton, Peter; Ramesar, RajkumarThe inherited retinal degenerative disorders are a common cause of severe visual handicap in the W estem world. Retinitis pigmentosa (RP) is a group of retinopathies in which a primary feature is a progressive loss of photoreceptor and retinal pigment epithelium function. Over the last decade, investigations into the patho-physiology of RP have identified numerous disease-causing genes and loci (for a current listing refer to the web site http://www.sph.uth.tmc.edu/Retnet/). A study of a South African family with an autosomal dominant form of RP (adRP) forms the basis of this dissertation. In this family, comprising 44 individuals, the first manifestation of visual disturbance is usually evident between 20 and 30 years of age. Subsequently, another South African adRP family, consisting of 25 members, was also incorporated into this investigation. Genetic linkage analysis facilitated the mapping of the disease phenotype in the two South African adRP families to a 10 cM interval on chromosome 17q22. This novel locus, designated RP17, is the eighth identified for adRP. Haplotype construction in the two kindreds, in conjunction with multipoint analyses subsequently fine mapped RP17 to a 1 cM region between microsatellite markers D17S1604 and D17S948. Although the two families are from ethnically diverse population groups, they share the same disease-associated haplotype spanning 12 cM, which suggests that the disorder may be caused by the same pathogenic mutation in the same gene. The positional cloning approach was utilised in an endeavour to identify the RP17 gene and an attempt was made to construct a physical map of the 1 cM critical region. A contig consisting of seven yeast artificial chromosome (YAC) clones was assembled using sequence-tagged-site (STS) content mapping. In order to close a gap in the YAC contig, a bacterial artificial chromosome (BAC) library was screened and the vectorette PCR technique was used to verify overlapping sequences. This contig should provide a useful tool for the purpose of isolating genes or transcription units within the RP17 critical interval. In this regard, purified YAC DNA was isolated using pulsed-field gel electrophoresis and the cDNA selection technique was employed to generate a transcription map. This approach was applied to YAC 75Ic12 using a foetal brain cDNA library, and two rounds of selection were performed to create a sub-library for enriched cDNAs derived from this clone. Screening for the presence of contaminating sequences in the 480 transformants revealed that (i) approximately 7% of the selected clones contain COT-1 DNA and (ii) none of the clones were contaminated with yeast AB1380 DNA. Ten randomly chosen clones were sequenced and subjected to BLASTN analysis, which revealed the presence of a 23 bp contaminant, known genes as well as novel transcripts. In order to optimise efforts to isolate the adRP gene, four positional candidates residing on 17q were screened for evidence implicating them in the adRP phenotype in the two 17q22-linked families. The genes investigated were: PDEG (gamma subunit of rod phosphodiesterase), TIMP2 (tissue inhibitor of metalloproteinases-2), PKCA (protein kinase C alpha) and retinal fascin. These candidates were chosen on the basis of (i) mapping to 17q, (ii) expression in the retina and/or (iii) potential involvement in the rod phototransduction pathway. Recombination events between the adRP locus and a single strand conformation polymorphism (SSCP) in PDEG, and a restriction fragment length polymorphism (RFLP) in TIMP2 provided evidence for the exclusion of these candidate genes. A novel SSCP detected in the promoter region of retinal fascin was genotyped in the two adRP families and showed a lack of co-segregation with the disease locus. Furthermore, direct DNA sequencing of the coding regions as well as the promoter region of retinal fascin in RP affected family members did not reveal any pathogenic mutations. In addition, data is provided which suggests that PKCA does not reside on any of the YACs and BACs encompassing the RP17 critical interval. This gene is therefore unlikely to be responsible for the adRP phenotype in the two RP17-linked families. Ultimately, the work reported in this thesis may contribute to the body of knowledge on inherited retinal degenerative disorders. Moreover, this investigation should provide the basis for further study of the aetiology of RP in all families linked to the RP17 locus on chromosome 17q22. The immediate application of these molecular findings is the potential for pre-symptomatic testing of at-risk members from the two adRP kindreds.
- ItemOpen AccessPsychological trauma and posttraumatic stress disorder in a South African birth cohort study(2015) Koen, Nastassja; Stein, Dan J; Ramesar, RajkumarPsychological trauma - including exposure to intimate partner violence (IPV) - is highly prevalent in South Africa, and may result in posttraumatic stress disorder (PTSD) in a subset of individuals. Pregnant women and new mothers are particularly vulnerable; and trauma exposure and PTSD in this sub-group may be associated with a number of adverse maternal-child sequelae including poor birth outcomes and impaired infant neurodevelopment. Risk factors for psychological trauma exposure, and for subsequent PTSD, are likely to include environmental and genetic influences. Given the high burden of trauma and related disorders, the unique genetic ancestry, and the relative paucity of empirical data, further work in South African populations is warranted. This thesis aimed to investigate a number of questions about trauma and PTSD in the Drakenstein Child Health Study (an ongoing South African birth cohort study), including their risk factors, their impact on infant birth anthropometry and development, and their genetic correlations. This thesis includes five publications, all presenting data from the Drakenstein Child Health Study. Pregnant women were recruited from two clinics in the Drakenstein sub-district - a peri-urban community outside Cape Town, Western Cape. Sociodemographic characteristics; psychosocial risk factors (including depression, stressful life events, psychological distress and alcohol and substance misuse); trauma exposure (childhood trauma, IPV and lifetime trauma); and PTSD were assessed using validated and reliable self-reported questionnaires, as well as diagnostic psychiatric interviews.
- ItemOpen AccessPXR and CAR single nucleotide polymorphisms influence plasma efavirenz levels in South African HIV/AIDS patients(BioMed Central Ltd, 2012) Swart, Marelize; Whitehorn, Heather; Ren, Yuan; Smith, Peter; Ramesar, Rajkumar; Dandara, ColletBACKGROUND: This study investigated variation in NR1I2 and NR1I3 and its effect on plasma efavirenz levels in HIV/AIDS patients. Variability in plasma drug levels has largely led research on identifying causative variants in drug metabolising enzyme (DME) genes, with little focus on the nuclear receptor genes NR1I2 and NR1I3, coding for PXR and CAR, respectively, that are involved in regulating DMEs. METHODS: 464 Bantu-speaking South Africans comprising of HIV/AIDS patients on efavirenz-based treatment (n=301) and 163 healthy subjects were genotyped for 6 SNPs in NR1I2 and NR1I3. 32 of the 301 patients had their DNA binding domains (DBDs) in NR1I2 and NR1I3 sequenced. RESULTS: Significantly decreased efavirenz plasma concentrations were observed in patients carrying the NR1I3 rs3003596C/C and T/C genotypes (P=0.015 and P=0.010, respectively). Sequencing resulted in the discovery of a further 13 SNPs, 3 of which are novel variants in the DBD of NR1I2. There were significant differences in the distribution of NR1I2 and NR1I3 SNPs between South Africans when compared to Caucasian, Asian and Yoruba population groups. CONCLUSION: For the realisation of personalised medicine, PXR and CAR genetic variation should be taken into consideration because of their involvement in the regulation of DMEs.