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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/49973 |
| Date | 12 1900 |
| Creators | Hendricks, Roshan |
| Contributors | Hayes, V. M., Heyns, C. F., Hillermann, R., Stellenbosch University. Faculty of Medicine & Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 214 p. : ill. |
| Rights | Stellenbosch University |
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