Colorectal cancer (CRC) is the second most common cancer in Hong Kong. While high-penetrance germline mutations account for up to 6% of cases, much of the variation in genetic risk may be attributable to multiple low-penetrance variants. Previous genome wide association studies (GWAS) have identified a number of CRC susceptibility alleles in Caucasian populations. Our GWAS investigated the association between genetic variants with CRC risk in the Han Chinese population in Hong Kong. In Stage I, genomic DNA samples from 455 female Chinese CRC subjects were genotyped using the Illumina 610 Quad SNP chip. Association analysis was performed on 439 cases and 771 general population female controls recruited for a study on bone mineral density. Population stratification was examined through principal components analysis using EIGENSTRAT version 2.0. From the association results, 46 SNPs (Group 1) were selected for follow-up replication (Stage II), together with 10 SNPs (Group 2) from previous GWAS studies. Genomic DNA samples from 3,571 Chinese subjects were genotyped using Sequenom MassARRAY system. Association analysis was performed on 1,505 cases and 1,452 controls. 5 SNPs (rs835378, rs2652007, rs2139273, rs2139273 and rs9286410) exceeded the genome-wide significance level in stage I, although none replicated in Stage 2, suggesting genotyping error. Results from stage II showed that the three most significant SNP were among those selected from the previous studies, yet their significance levels in Stage I were very weak . None of the SNPs selected from Stage I was significant at p<0.01 in Stage 2. Two composite scores of genetic susceptibility, one for each group of SNPs, were calculated in stage II genotype data, as the total number of high-risk alleles (according to the direction of effect in Stage I results or previous GWAS) present in an individual. Both composite scores were significantly associated with CRC risk in Stage 2 (Group 1, p=2.38 x 10-5, beta=0.046, SE=0.012; Group 2 p=1.06 x 10-7, beta=0.10, SE=0.019), suggesting that while we had insufficient power to confirm individual SNPs identified in our GWAS and the previous GWAS, these findings indicate that the SNP sets selected from Stage I results, as well as those selected from previous GWAS, contain SNPs with genuine effects on CRC risk. One SNP, rs10795668 (OR = 0.79 [CI] 95%:0.71 – 0.87 p=3.78 x 10-6), was significantly associated with CRC risk in Stage II after adjustment for multiple testing. Two further SNPs, rs6983267 and rs4939827, also achieved suggestive p-values in Stage II. All these SNPs were selected from previous GWAS in the Caucasian population, demonstrating that shared genetic factors operate for CRC in diverse populations. / published_or_final_version / Psychiatry / Master / Master of Philosophy

Polycomb group (PcG) proteins governs the regulation of diverse cellular functions, such as cell fate decision, cell cycle progression, maintenance of embryonic stem cell pluripotency, and DNA damage repair. Although aberrant expression of PcG proteins has been frequently reported in different cancer types, CBX8 is one of the least studied PcG family members in cancer. Recently, a study showed that forced expression of CBX8 in normal human and mouse fibroblasts demonstrated that cells could bypass senescence via INK4a-ARF repression; while another report demonstrated that CBX8 was involved in MLL-AF9-linked leukemogenesis. Despite accumulating evidence on CBX8-related carcinogenic functions, the role of CBX8 in solid cancers has not been investigated thus far. This study is therefore initiated to investigate and establish the functional role of CBX8 in colorectal cancer. In this study, expression of CBX8 in 121 pairs of human CRC samples was analyzed by immunohistochemistry; and data were correlated with different clinicopathological parameters. To evaluate the functional effects of CBX8, CBX8 overexpressed and downregulated clones were established from three CRC cell lines. The in vitro effects of CBX8 on cell proliferation, cell cycle progression and apoptosis profiles were investigated; and the effects of CBX8 on tumorigenicity in vivo were further demonstrated in mice xenograft models. The results showed that CBX8 expression was downregulated or loss in approximately 48.8% of human colorectal tumors, and downregulated or loss of CBX8 expression were mainly observed in tumors with intermediate to later stages (stage II to IV). Moreover, expression of CBX8 showed a significant inverse correlation with colorectal tumor sizes (P < 0.0001). Ectopic expression of CBX8 in CRC cell lines resulted in inhibition of cell proliferation, clonogenic ability and anchorage-independent growth, which are hallmarks of tumorigenesis. Conversely, downregulation of CBX8 promoted proliferation and clonogenic ability. Moreover, it was found that restoring CBX8 expression could induce G0/G1 arrest of cell cycle. The tumor suppressive role of CBX8 in colorectal cells was further demonstrated in vivo through subcutaneous and orthotropic mice tumor models; followed by immuno-staining of the proliferation marker Ki-67. To unveil the possible mechanisms behind the tumor suppressing effects of CBX8, two signalling pathways commonly engaged in CRC were evaluated. At least part of the effects could be attributed to the mediation of MAPK signaling pathway; whereas the Wnt signalling was not affected by CBX8. This study demonstrated for the first time the loss of CBX8 expression in intermediate and late stage tumors, and was the first to report the tumor suppressing ability of CBX8 in solid cancers. The effects of CBX8 in this study were different to the functional implications reported in the current literature. This functional divergence in distinct cell types suggested a dynamic role of CBX8 depending on specific cellular context. / published_or_final_version / Surgery / Master / Master of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy

published_or_final_version / Surgery / Master / Master of Philosophy

Heterozygous mutations in BRCA1 or BRCA2 predispose carriers to an increased risk for breast or ovarian cancer. Both BRCA1 and BRCA2 (BRCA1/2) play an integral role in promoting genomic stability through their respective actions during homologous recombination (HR) mediated repair and stalled replication fork protection from nucleolytic degradation. SMARCAD1 (SD1) is a SWI/SNF chromatin remodeler that has been implicated in promoting long-range end resection and contributes to HR. Using human cell lines, we show that SMARCAD1 promotes nucleolytic degradation in BRCA1/2-deficient cells dependent on its chromatin remodeling activity. Moreover, SMARCAD1 prevents DNA break formation and promotes fork restart at stalled replication forks. These studies identify a new role for SMARCAD1 at the replication fork. In addition to the work presented here, I discuss a method for introducing stop codons (nonsense mutations) into genes using CRISPR-mediated base editing, called iSTOP, and provide an online resource for accessing the sequence of iSTOP sgRNASs (sgSTOPs) for five base editor variants (VQR-BE3, EQR-BE3, VRER-BE3, SaBE3, and SaKKH-BE3) in humans and over 3 million targetable gene coordinates for eight eukaryotic species. Ultimately, with improvements to CRISPR base editors this method can help model and study nonsense mutations in human disease.

Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Prostate cancer (CaP) has the highest incidence of any malignancy affecting South African males. The aetiology of prostate carcinoma indicate that ethnicity is one of the most important risk factors. The causes of these ethnic differences are unknown but presumably involve both environmental and genetic factors. Carcinoma of the prostate is androgen dependent, and it has been suggested that variations in androgen metabolism and synthesis may affect an individuals' risk. Therefore, genes involved in these pathways are candidates for determining CaP susceptibility. In this study two candidate genes in the androgen biosynthetic and metabolic pathway were analysed, viz., the androgen receptor gene (AR), involved in androgen transport and transcriptional activation, and the cytochrome p450c17a gene (CYP17), important for testosterone biosynthesis. Comprehensive mutation detection assays were designed (appropriate for analysis of archival paraffin-embedded material) for almost the entire coding region (excluding polymorphic repeat sequences), and including all splice site junctions of the AR gene, as well as the entire coding region of CYP17. The aim of this study was thus to determine the type and frequencies of genetic variants of these androgen metabolism genes within the diverse South African population, and to determine if the observed ethnic variation in the incidence and progression of CaP can be explained by ethnic-based genetic differences. For high sensitivity mutation detection, the most powerful of the pre-screening methods was used, namely denaturing gradient gel electrophoresis (DGGE). 20 CaP and 25 control benign prostatic hyperplasia (BPH) tissue samples were screened in order to identify possible mutations. Blood samples from the same patients were analysed in order to determine whether mutations are germline and therefore present in all cells of the body. Additional blood samples from the Western Province Blood Transfusion Service (WPBTS) (Refer to section 2.1.2, Table) were also analysed in order to determine the frequency of identified polymorphisms within the general population. Certain polymorphisms were further analysed in paraffin-embedded wax material (exclusively from Blacks) to determine the distribution of these polymorphisms in the Black population. Direct sequencing of mutant-containing DNA fragments was performed to determine the exact location and nature of mutation. Using the AR- DGGE assay 4 novel mutations were identified as well as a previously reported codon 211 (E211) polymorphism. With the CYP17- DGGE assay, 3 novel single nucleotide polymorphisms (SNPs) were detected. Three base variants occured, in codons 36 (L36), 46 (H46) and 65 (S65), as well as intronic substitutions in intron 4 (IVS+58G4C) and intron 6 (IVS-25C7A). Frequencies of SNPs were measured in the CaP and BPH samples. In conclusion, the identified polymorphisms could be used as markers in determining CaP susceptibility and may thus facilitate the identification of individuals with a high- or low-risk of developing carcinoma of the prostate. / AFRIKAANSE OPSOMMING: Prostaatkanker vertoon die hoogste voorkoms van enige kwaardaardigheid wat Suid-Afrikaanse mans aantas. Die etiologie van prostaatkarsinoom dui aan dat etnisiteit een van die mees belangrike risikofaktore is. Oorsake van hierdie etniese verskille is onbekend, maar vermoedelik is omgewing en genetiese faktore albei betrokke. Karsinoom van die prostaat is androgeenafhanklik en daar is voorgestel dat variasies in androgeenmetabolisme en androgeensintese 'n persoon se risiko mag affekteer. Gevolglik, is gene betrokke in hierdie paaie kandidate vir die bepaling van prostaatkanker vatbaarheid. In hierdie studie het ons twee kandidaat gene in die androgeen biosintetiese en metaboliese pad geanaliseer, naamlik, die androgeen reseptor geen (AR), betrokke in androgeen vervoer en aktivering van transkripsie, en die sitokroom p450c17a geen (CYP17), belangrik vir testosteroon biosintese. Ons het omvattende mutasie-bespeurings-essai-sisteme ontwikkel (ook uitvoerbaar op argivale paraffien-bewaarde materiaal), wat amper vir die hele koderende streek van die AR geen gebruik kan word (uitsluitend herhalende polimorfiese reekse) en wat alle splytpunt-aansluitings van die AR geen insluit, asook vir die hele koderende streek van CYP17. Die doel van hierdie studie was dus om die tipe en frekwensies van genetiese variante van androgeen metabolisme gene in ons diverse Suid-Afrikaanse bevolking te bepaal, en om vas te stel of die waarneembare etniese wisseling in die insidensie en vordering van prostaatkanker verstaan kan word deur etnies gebaseerde genetiese verskille. Die mees sensitiewe tegniek wat tans beskikbaar is vir vooraf-sifting vir onbekende mutasies is gekies, naamlik denaturerende gradiënt gel elektroforese (DGGE). Om moontlike mutasies op te spoor, het ons 20 prostaatkanker en 25 benijne prostaathiperplasie (BPH) monsters geanaliseer. Analise was gedoen op bloedmonsters van dieselfde pasiënte om vas te stel of kiemlyn mutasies (in alle liggaamselle) teenwoordig is. Bykomstige bloedmonsters (van die Westelike Provinsie Bloedoortappingsdiens) is ook geanaliseer om die frekwensie van bespeurde polimorfismes in die algemene bevolking te bepaal. Argivale paraffien-bewaarde materiaal (eksklusief van Swartes) is ook geanaliseer om die verspreiding van sekere polimorfismes in die Swart bevolking te bepaal. Direkte DNA volgorde bepaling van mutante DNA fragmente is uitgevoer om die ligging en tipe van mutasies te bepaal. Met die toepassing van ons AR-DGGE mutasiesisteem het ons 4 nuwe mutasies ontdek asook 'n kodon 211 (E211) polimorfisme wat voorheen gevind is. Vyf enkel nukleotied polimorfismes is met die CYP17-DGGE mutasiesisteem opgespoor. Die polimorfismes sluit in: drie basis veranderinge wat voorkom in kodons 36 (L36), 46 (H46) en 65 (S65), asook introniese substitutisies in intron 4 (IVS+58G4C) en intron 6 (IVS-25C7 A). Frekwensies van die polimorfismes was bereken in die prostaatkanker en BPH monsters. Die resultate aangebied in hierdie tesis dui aan dat die gevonde polimorfismes as merkers gebruik kan word om prostaatkanker vatbaarheid te bepaal en daardeur individue te identifiseer met 'n hoë of lae risiko vir prostaatkarsinoom ontwikkeling.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Pathology / Master / Master of Philosophy