Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: The work presented in this thesis deals with the association between cancers of
the upper gastro-intestinal tract and the tumor suppressor gene, TP53, and can
be divided into three parts: (i) the analysis of the mutational spectrum of TP53
with respect to laryngeal cancer, (ii) the analysis of the mutational spectrum of
TP53 with respect to esophageal cancer and (iii) the analysis of TP53
transcriptional levels in esophageal cancer.
Laryngeal cancer (LC) is the 6th most common cancer in the world and the 2nd
most common respiratory cancer, with approximately 500 000 new cases per
annum detected worldwide. Over the last few years, LC has become
increasingly prevalent within the Coloured Community of the Western Cape. The
mechanisms of tumorigenesis in LC remain unknown, although smoking and
alcohol consumption are considered to be major risk factors. Mutations within
the gene TP53 have been strongly implicated as playing a role in cancer
development, as they are frequently found in several cancer types. We therefore
screened exons 5 - 8 of TP53 for mutations in DNA from tumor biopsies (n=44)
and blood samples (n=42) from Coloured LC patients, using polymerase chain
reaction - single strand conformation polymorphism (PCR-SSCP) analysis and
direct sequencing. Blood samples from a healthy, matched control group (n=40)
were included in the study as controls. Significant correlations were found
between the occurrence of LC and age and smoking, whereas daily meat
consumption was a possible protective factor. In tumor-derived samples,
mutations were found in 3 of the exons under investigation, representing 25% of
the samples. The mutations were unique to the tumor biopsies, indicating a
somatic origin for mutations. The data confirms that the region between codons
175 and 273 of TP53 is a mutational hotspot for cancers in general. This study
reports 6 novel mutations within this same region. Esophageal cancer (EC) has a very high incidence in South Africa, relative to the
rest of the world, and is particularly common amongst the Black Transkei
population. The goal of this study was to determine whether there are
differences in the TP53 mutational pattern observed in the Coloured Western
Cape community as compared to that observed in the Black Transkei community.
This required the analysis of the molecular structure of TP53, specifically exons 5
- 8, in a group of Coloured EC patients (n=44) treated at Tygerberg Hospital,
Cape Town, South Africa. DNA obtained from tumor biopsies and blood (from
patients) as well as from apparently healthy surrounding tissue was screened via
PCR-SSCP and direct sequencing analysis. Only 4 nucleotide changes were
observed from a total of 124 sequences obtained, of which two were novel to
esophageal squamous cell carcinoma. These 4 nucleotide alterations were
found only within the tumor biopsy sample set, representing 9% of the tumors
investigated. This study revealed that the mutational spectrum of TP53 within
the Coloured population of the Western Cape greatly differs from that of the
Black community of the Transkei. This suggests that a different set of etiological
factors are involved in the tumorigenic process for each of these distinct
geographical communities, which is the subject of an epidemiological study
undertaken by the MRC.
The final part of this thesis deals with the quantification and comparison of TP53
transcription levels in esophageal cancer tumor tissue to the TP53 levels in
healthy esophageal tissue obtained from patients from a unique geographical
and ethnic background. The cohort used in this study consisted of Coloured
patients (n=2) treated at Tygerberg Hospital. The LightCycler system was
implemented in order to try to accurately quantify TP53 mRNA levels.
Unfortunately, the desired results were unattainable due to unforeseen difficulties
encountered during the study. These difficulties included the insufficient
preservation of samples for RNA based studies. Several recommendations were
made concerning future similar studies, including an improved planning strategy
as well as the employment of an RNA stabilizing agent. Additionally, a few important contributions were made through this study, including the design and
optimization of TP53 primers specifically intended for future RNA studies. These
primers would enable the identification of the presence of TP53 RNA species as
well as the absence of DNA contamination in a single PCR amplification step.
Other contributions include the development of a well-optimized RNA extraction
method for the extraction of RNA from tough tissues (such as the human
esophageal tissue used in this study). This method makes the extraction of large
quantities of RNA from small amounts of tough tissue types possible.
In conclusion, this study has made a significant contribution to the field of cancer
research, by shedding light on the TP53 mutational spectrum with regards to
laryngeal as well as esophageal cancer in a population unique to the Western
Cape.
The first part of this thesis has been published in Cancer Genetics and
Cytogenetics (Barnard, D., K. Lehmann, E.G. Haal, P.O. van Heiden, and l.C.
Victor. 2003. The spectrum of mutations in TP53 in laryngeal cancer patients
from a high-incidence population shows similarities to many of the known
mutational hotspots. Cancer Genetics and Cytogenetics 145:126-132), of which
a copy can be found in Appendix I. This work has also been presented (by D.
Barnard) at an international conference entitled "Cancer of the Esophagus and
Gastric Cardia: From Gene to Cure", held in Amsterdam, the Netherlands during
the period 13 - 15 December 2002. / AFRIKAANSE OPSOMMING: Die werk wat in hierdie tesis voorgelê word handel oor die assosiasie tussen
kankers van die boonste gastrointestinale weg en die tumor suppressor geen,
TP53, en kan in 3 dele gedeel word, (i) die analise van die mutasiespektrum van
TP53 in laringiale kanker (LK), (ii) die analise van die mutasiespektrum van TP53
in slukderm kanker (SK) en (iii) die analise van die transkripsievlakke van TP53
in SK.
Laringeal kanker (LK) is die 6de algemeenste kanker in die wêreld en die 2de
algemeenste respiratoriese kanker, met "n benaderde 500 000 nuwe gevalle
jaarliks wêreldwyd. Oor die afgelope paar jare het LK "n toenemende probleem
geraak, veral in die Kleurling gemeenskap van die Wes Kaap. Die meganismes
van die tumorvorming in LK is onbekend, alhoewel rook-en alkoholgebruik
vername risiko faktore is. Die voorkoms van mutasies in TP53 is verskeie kere
aangetoon in verskillende kanker tipes en daar word vermoed dat dit "n rol speel
in tumorvorming. In hierdie studie is dus na mutasies in eksons 5 - 8 van TP53
gesoek in tumor biopsie weefsel (n=44) en bloed isolate (n=42) van Kleurling LK
pasiënte d.m.v. polimerase ketting reaksie - enkelstring konformasie
polimorfisme (PKR-ESKP) analisering en direkte volgorde bepaling. Bloed
monsters van "n vergelykbare groep (n=40) is ook in die studie ingesluit as "n
kontrole. Betekenisvolle positiewe korrelasies is gevind tussen die voorkoms van
LK en ouderdom sowel as rook. Daarmee saam is daaglikse vleisinname as
potensiële beskermende faktor gevind. In tumor biopsies is mutasies in 3 van
die ondersoekte eksons gevind, wat 25% van die biopsie monsters
verteenwoordig. Hierdie mutasies is uniek aan die tumor biopsie weefsels en dui
op "n somatiese oorsprong van mutasies. Hierdie bevindinge bevestig dat die
gedeelte tussen kodons 173 - 273 van TP53 "n hipermuteerbare gebied
geassosieer met kankers is. Hierdie studie bevestig 6 nuwe mutasies.
Daar is 'n hoë insidensie van slukderm kanker (SK) in Suid Afrika relatief tot die
res van die wêreld. Hierdie soort kanker word veral gevind by die Swart
populasie van die Transkei. Die doel van hierdie studie was om verskille tussen
die TP53 mutasie patroon van die Kleurling gemeenskap van die Wes Kaap en
die Swart gemeenskap van die Transkei te vergelyk. Hiervoor is die molekulêre
struktuur van TP53, veral eksons 5 - 8, in 'n groep Kleurling SK pasiënte (n=42)
wat behandel is by Tygerberg Hospitaal, Kaapstad, Suid Afrika, geanaliseer.
Analisering is gedoen deur DNS van tumor, bloed en ook oënskynlike gesonde
aangrensende weefsel van dieselfde pasiënte te onderwerp aan PKR-ESKP
analise en direkte volgorde bepaling. Slegs 4 nukleotied veranderings is gevind
in 124 volgorde bepalings, waarvan 2 nuwe veranderings is in SK. Hierdie 4
nukleotied veranderinge verteenwoordig 9% van al die tumors wat ondersoek is
in die studie. Hierdie studie bewys dat die mutasiespektrum van TP53 in die
Kleurling gemeenskap van die Wes Kaap grootliks verskil van die Swart
gemeenskap van die Transkei. Dit impliseer dat verskillende etiologiese faktore
moontlik 'n rol mag speel op die tumorvormingsproses in die 2 afsonderlike
geografiese gemeenskappe. Hierdie is die onderwerp van 'n epidemiologiese
studie wat deur die MNR onderneem word.
Die laaste deel van hierdie tesis handel oor die kwantifisering en vergelyking van
TP53 transkripsievlakke in SK tumor weefsel teenoor TP53 vlakke in gesonde
slukderm weefsel van pasiënte in 'n unieke geografiese en etniese agtergrond.
Die studie populasie in hierdie projek het bestaan uit Kleurling pasiënte (n=2) wat
by Tygerberg hospitaal behandel is. Die "LightCycler" sisteem is gebruik vir die
akkurate kwantifisering van TP53 boodskapper RNS vlakke. Ongelukkig is die
verlangde resultate nie gekry nie as gevolg van onvoorsiene probleme wat
ondervind is tydens die studie. Hierdie probleme sluit in die onvoldoende
preserv RNS studies. Hierdie inleiers maak dit nou moontlik om die teenwoordigheid van
TP53 RNS spesies sowel as die afwesigheid van DNS kontaminasie in een PKR
amplifikasie stap te kan identifiseer. 'n Ander belangrike bydrae is die
ontwikkeling van 'n goed geoptimaliseerde RNS ekstraksie metode vir moeilike
starre weelfsel tipes (soos menslike slukderm weefsel in hierdie studie) en maak
die ekstraksie van groot hoeveelhede RNS uit klein hoeveelhede van moeilik
hanteerbare weefsel tipes moontlik.
Om saam te vat, hierdie studie het betekenisvolle bydraes gemaak tot die veld
van kankernavorsing deur die ontrafeling van die TP53 mutasiespektrum in beide
laringeale sowel as slukderm kanker, in 'n populasie uniek aan die Wes Kaap.
Die eerste deel van hierdie tesis is gepubliseer in Cancer Geneties and
Cytogenetics (Barnard, D., K. Lehmann, E. G. Hoal, P. D. van Heiden, and T. C.
Victor. 2003. The spectrum of mutations in TP53 in laryngeal cancer patients
from a high-incidence population shows similarites to many of the known
mutational hotspots. Cancer Genetics and Cytogenetics 145: 126-132) en 'n
afskrif van die artikel is ingesluit in Appendix I. Hierdie werk is ook voorgedra
(deur D. Barnard) by 'n internasionale kongres getiteld "Cancer of the
Esophagus and Gastric Cardia: From Gene to Cure", wat in Amsterdam,
Nederland gehou is gedurende 13 - 15 Desember 2002
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/50046 |
| Date | 03 1900 |
| Creators | Barnard, Desire |
| Contributors | Victor, T. C., 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 | 195, [8] p. : ill. |
| Rights | Stellenbosch University |
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