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ETRESIA VAN DYK (M.Sc.)
12126497
Thesis submitted for the degree Doctor of Philosophy in Biochemistry at the
Potchefstroom campus of the North-West University
Promoter:
July 2011
Prof P.J. Pretorius
GENADE, ALLES NET GENADE
-Totius
Abstract
Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder of the tyrosine
degradation pathway.
The defective fumarylacetoacetate hydrolase enzyme causes the
accumulation of upstream metabolites such as fumarylacetoacetate (FAA), maleylacetoacetate
(MAA), succinylacetone (SA) and p-hydroxyphenylpyruvic acid (pHPPA).
In vitro and in vivo
studies showed that the accumulation of these metabolites are detrimental to cell homeostasis, by
inducing cell cycle arrest, apoptosis, and endoplasmic reticulum stress, depleting GSH, inhibiting
DNA ligase, causing chromosomal instability, etc. For in vivo studies different models of HT1 were
developed.
Most notably was the fah deficient mouse, whose neonatally lethal phenotype is
rescued by the administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC).
Although, this model most closely resembles the human phenotype with elevated tyrosine levels
and the development of hepatocellular carcinoma (HCC), the model is not human genome based.
Both the in vitro and in vivo studies suggested that DNA repair is affected in HT1.
However, it is not yet clear which DNA repair mechanisms are affected and if only protein
functionality is affected, or if expression of DNA repair proteins are also affected.
Characteristic of HT1 is the high prevalence of HCC and the presence of liver mosaicism.
The liver mosaicism observed in HT1 patients are the result of reversion of the inherited mutation
to wild-type. The general consensus is that the reversion is the result of a true back mutation.
However, the mechanism underlying the back mutation is still unresolved.
It was suggested that cancer develops either through a chromosomal instability mutator
phenotype, a microsatellite instability mutator phenotype, or a point mutation instability mutator
phenotype. In HT1 only chromosomal instability was reported.
The aims of this study were to contribute to the understanding of the molecular basis of the
genetic mosaicism in hereditary tyrosinemia type 1. More specifically, determine whether baseand nucleotide DNA repair mechanisms are affected and to what extent, and to determine if
microsatellite instability is found in HT1. To achieve these aims, a parallel approach was followed:
i.e. to develop a HT1 hepatic cell model and to use HT1 related models and HT1 patient material.
To assess the molecular basis of the genetic mosaicism in HT1, the comet assay, gene expression
assays, microsatellite instability assays, high resolution melting and dideoxy sequencing
techniques were employed.
i
ABSTRACT
Results from the comet assay showed that the HT1 accumulating metabolites, SA and
pHPPA, decreased the capacity of cells for base- and nucleotide excision repair. Gene expression
assays showed that short term exposure to SA and/or pHPPA do not affect expression of hOGG1
or ERCC1.
The expression of these genes were, however, low in HT1 patient samples.
Microsatellite instability assays showed allelic imbalance on chromosome 7 of the mouse genome,
and microsatellite instability in the lymphocytes of HT1 patients. Although high resolution melt and
sequencing results did not reveal any de novo mutations in fah or hprt1, the appearance of de
novo mutations on other parts of the genome can not be ruled out.
To conclude, results presented in this thesis, for the first time show that in HT1 the initiating
proteins of the base- and nucleotide repair mechanisms are affected, the gene expression of DNA
repair proteins are low, and microsatellite instability is found in HT1.
By contributing to the
elucidation of the mechanism underlying the development of HT1-associated HCC, and providing
evidence for the development of a mutator phenotype, the results presented in this thesis
contributes to the understanding of the molecular mechanisms underlying the genetic mosaicism in
HT1.
In addition to these contributions, a hypothesis is posited, which suggests that a point
mutation instability (PIN) mutator phenotype is the mechanism underlying the mutation reversions
seen in HT1.
ii
Samevatting
Die onderwerp van hierdie studie is: “Die molekulêre agtergrond van die genetiese mosaïek
in oorerflike tirosinemie (HT1)”. Oorerflike tirosinemie tipe 1 (HT1) is 'n outosomale resessiewe
versteuring van die tirosien-katabolisme. Die defektiewe fumarielasetoasetaat-hidrolase ensiem
veroorsaak
dat
intermediêre
metaboliete
soos
fumarylasetoasetaat,
suksinielasetoon (SA) en p-hidroksiefenielpiruvaat (pHPPA) ophoop.
maleïelasetoasetaat,
Beide in vitro en in vivo
studies het getoon dat die opeenhoping van hierdie metaboliete nadelig is vir sel-homeostase deur
onder andere staking van die selsiklus, apoptose, en endoplasmiese retikulum stres te induseer,
glutatioonvlakke uit te put, DNS-ligase te inhibeer en chromosomale-onstabiliteit te veroorsaak. Vir
in vivo studies is verskillende HT1-modelle ontwikkel, waarvan die belangrikste die fah-gebrekkige
muis is, en die neonatale fenotipe van hierdie model word grootliks beskerm deur behandeling met
NTBC.
Alhoewel hierdie model baie ooreenstem met die menslike fenotipe, met verhoogde
tirosien-vlakke en die ontwikkeling van HCC, is die model nie op die menslike genoom gebaseer
nie.
Beide in vitro en in vivo studies dui daarop dat DNS-herstel benadeel word in HT1, maar dit
is egter nog nie duidelik watter DNS-herstelmeganismes geraak word nie, en of slegs
proteïenfunksie, of uitdrukking van DNS-herstelproteïene ook geraak word nie.
Kenmerkend van HT1 is die hoë voorkoms van hepatosellulêre karsinoom (HCC) en die
teenwoordigheid van ‘n lewermosaïek. Die lewermosaïek wat in HT1 pasiënte waargeneem word,
is die gevolg van die omkeer van die oorgeërfde mutasie tot die wilde-tipe.
konsensus is dat hierdie omkeer die resultaat is van 'n ware terugmutasie.
Die algemene
Die meganisme
onderliggend hieraan is egter nog onbekend.
Die algemene oortuiging is dat kanker kan ontwikkel óf deur ‘n chromosomale-onstabiliteit
gegenereerde mutasie-fenotipe óf 'n mikrosatelliet-onstabiliteit genereerde mutasie-fenotipe. In
HT1 is daar al chromosomale-onstabiliteit waargeneem, maar nie mikrosatelliet-onstabiliteit nie.
Die doel van hierdie studie was om by te dra tot die ontrafeling van die molekulêre basis
van die genetiese mosaïek wat voorkom in tirosinemie tipe 1. Meer spesifiek, om vas te stel of
basis- en nukleotied DNS-herstelmeganismes beïnvloed word en tot in watter mate, en om te
bepaal of mikrosatelliet-onstabiliteit in HT1 waargeneem word. Om hierdie doelwitte te bereik is 'n
parallelle benadering gevolg. Aan die een kant is 'n HT1 hepatiese sel model ontwikkel deur RNStussenkoms tegnologie, en aan die ander kant is van HT1-verwante modelle en HT1-
iii
SAMEVATTING
pasiëntmateriaal gebruik gemaak. Om te molekulêre basis van die genetiese mosaïek in HT1 te
bepaal is die volgende analises uitgevoer: die komeet analise, geenuitdrukking analise,
mikrosatelliet-onstabiliteit analise, hoë resolusie smelt, en volgordebepalings.
Resultate van die komeet analise het getoon dat die HT1-akkumulerende metaboliete, SA
en pHPPA, die vermoë van selle vir basis- en nukleotied DNS-herstel verlaag. Geenuitdrukkings
analises het getoon dat die korttermyn blootstelling aan SA en/of pHPPA nie die uitdrukking van
hOGG1 of ERCC1 beïnvloed nie. Die uitdrukking van hierdie gene is egter aansienlik verlaag in
HT1-pasiëntmateriaal. Mikrosatelliet-onstabiliteit analises het getoon dat alleliese-wanbalans in
chromosoom 7 van die muis genoom, en mikrosatelliet-onstabiliteit in die limfosiete van HT1pasiënte voorkom. Alhoewel in die hoë resolusie smelt en volgordebepalings resultate de novo
mutasies in fah of hprt1 nie waargeneem is nie, kan die voorkoms van de novo mutasies in ander
dele van die genoom nie uitgesluit word nie.
Die resultate wat in hierdie proefskrif vervat is, wys vir die eerste keer dat in HT1, basis- en
nukleotied DNS-herstelmeganismes geraak word, die geen uitdrukking van DNS-herstelproteïene
verlaag is, en dat mikrosatelliet-onstabiliteit in HT1 waargeneem word. Deur by te dra tot die
toeligting van die meganismes onderliggend aan die ontwikkeling van HT1-verwante HCC, en die
verskaffing van bewyse vir die ontwikkeling van 'n mutasiegenererende fenotipe, dra die resultate
by tot die begrip van die molekulêre meganismes onderliggend aan die genetiese mosaïek gesien
eie aan HT1. Bo en behalwe vir hierdie bydraes, is 'n hipotese gemaak dat ‘n puntmutasieonstabiliteit (PIN) mutasiegenererende fenotipe die meganisme onderliggend is aan die mutasie
omkerings wat in HT1 mag plaasvind.
iv
Keywords
Hereditary tyrosinemia type1; Mosaicism; Hepatocellular carcinoma; DNA repair; Genome
instability; Mutator phenotype
v
Table of content
Abstract ...................................................................................................................... i
Samevatting .............................................................................................................. iii
Keywords .................................................................................................................. v
Table of content ........................................................................................................ vi
List of symbols ........................................................................................................ viii
List of abbreviations ................................................................................................... ix
List of figures .......................................................................................................... xiv
List of tables ............................................................................................................ xv
Chapter 1: Introduction.............................................................................................. 1
Chapter 2: Literature review....................................................................................... 4
2.1 Hereditary tyrosinemia type 1 ................................................................................. 4
2.1.1 Introduction to inherited metabolic disorders .................................................... 4
2.1.2 Inherited disorders of the tyrosine catabolism .................................................. 5
2.1.3 Hereditary tyrosinemia type 1........................................................................... 7
2.1.3.1 General considerations ............................................................................. 7
2.1.3.2 Diagnosis and treatment ..........................................................................10
2.1.3.3 Genetics of FAH .......................................................................................10
2.1.3.4 Mutation reversion ....................................................................................11
2.1.3.5 Accumulating metabolites.........................................................................12
2.1.3.6 HT1 models ..............................................................................................14
2.2 Mosaicism..............................................................................................................15
2.2.1 Introduction to mosaicism ...............................................................................15
2.2.2 Mosaicism in HT1 ...........................................................................................16
2.3 DNA repair .............................................................................................................17
2.3.1 Introduction to DNA repair mechanisms ..........................................................17
2.3.2 DNA excision repair mechanisms....................................................................17
2.3.2.1 Base excision repair .................................................................................18
2.3.2.2 Nucleotide excision repair ........................................................................18
2.3.2.3 Mismatch repair (MMR) ............................................................................19
2.3.3 Consequences of defective DNA repair ..........................................................19
2.4 Summary ...............................................................................................................20
Chapter 3: HT1 hepatic cell model ........................................................................... 23
3.1 Development of a HT1 hepatic cell model ..............................................................23
3.1.1 shRNA oligonucleotide design, acquisition and annealing ...............................24
vi
TABLE OF CONTENT
3.1.2 Cloning shRNAs into pSIREN-RetroQ-Tet vector ............................................25
3.1.3 Pilot experiments ............................................................................................26
3.1.4 Development of a single-stable ptTS expressing cell line ................................26
3.1.5 Developing a double-stable cell line ................................................................27
3.2 Future considerations ............................................................................................33
Chapter 4: Methodology .......................................................................................... 35
4.1 Cell cultures .......................................................................................................35
4.2 DNA and RNA samples......................................................................................35
4.3 Comet assay modified for BER and NER ...........................................................36
4.4 Gene expression ................................................................................................37
4.4.1 RT-PCR ......................................................................................................37
4.4.2 Gene expression .........................................................................................38
4.5 Microsatellite analysis with Agilent 2100 Bioanalyzer .........................................39
4.6 High resolution melting.......................................................................................40
4.7 PCR and sequencing .........................................................................................41
Chapter 5: Results and discussion ........................................................................... 44
5.1
5.2
5.3
5.4
DNA damage and repair ....................................................................................44
Gene expression profiles ...................................................................................46
Genome stability ................................................................................................53
Point mutation instability ....................................................................................61
Chapter 6: Paper 1 ................................................................................................. 69
Chapter 7: Paper 2 ................................................................................................. 75
Chapter 8: Summary and conclusion ........................................................................ 82
Chapter 9: Paper 3 ................................................................................................. 88
References ............................................................................................................. 94
Appendix A: Vector maps ...................................................................................... 108
Appendix B ........................................................................................................... 110
Appendix C ........................................................................................................... 111
Appendix D ........................................................................................................... 112
Appendix E: Conference Abstract........................................................................... 114
vii
List of symbols
°C:
Degrees Celsius
µl:
Microlitre
µM:
Micromolar
Į:
Alpha
ȕ:
Beta
ǻ or į:
Delta
İ:
Epsilon
%:
Percentage
ij:
Phi
viii
List of abbreviations
A:
A:
Adenine
ANON:
Anonymous
ANOVA:
Analysis of variance
AP:
Abasic site
APC:
Adenomatous polyposis coli
ATCC:
American tissue culture company
ATP:
Adenosine triphosphate
B:
BER:
Base excision repair
BiP:
Binding immunoglobulin protein
bp:
Basepair
BRCA1:
Breast cancer 1
BSA:
Bovine serum albumin
C:
C:
Cytosine
CAF:
Central Analytical Facilty
cDNA:
Complementary DNA
CHOP:
CEBP homologous protein
CIN:
Chromosomal instability
cM:
Centi-Morgan
COLD-PCR:
Co-amplification at lower denaturation temperature-PCR
CSA:
Cockayne syndrome group A
CSB:
Cockayne syndrome group B
Cu:
Copper
D:
DDB:
DNA damage binding protein
ddH2O:
Double distilled water
DMEM:
Dulbecco’s modified eagles medium
DMSO:
Dimethylsulfoxide
DNA:
Deoxyribonucleic acid
DNMT1:
DNA methyltransferase 1
dNTP:
Deoxyribonucleotide triphosphate
DOPA:
Dihydroxyphenylalanine
ix
LIST OF ABBREVIATIONS
DOVA:
4,5-Dioxovaleric acid
Dox:
Doxycycline
DSB’s:
Double strand breaks
DTT:
Dithiothreitol
E:
EDTA:
Ethylenediamine tetraacetic acid
EGTA:
Ethylene glycol-bis(ȕ-aminoethyl ether) N,N,N’,N’-tetraacetic acid
eIF2Į:
Eukaryotic translation factor 2 alpha
ENU:
N-ethyl-N-nitrosourea
ERCC1:
Excision repair cross-complementing rodent repair deficiency, complementation group 1
ERK:
Extra cellular signal-regulated protein kinase
Et al:
Et Alii/Alia (Latin: And others)
EtBr:
Ethidium Bromide
EtOH:
Ethanol
EXO1:
Exonuclease 1
F:
FA:
Fumarylacetone
FAA:
Fumarylacetoacetate
FAH:
Fumarylacetoacetate hydrolase
FBS:
Fetal bovine serum
Fe:
Iron
FPG:
Formamido-pyrimidine glycosylase
Fwd:
Forward
G:
g:
Gram
g:
Gravitational force
G:
Guanine
Gas:
Growth arrest specific 2
GGR:
Global genomic repair
GRP17:
Gadd-related protein 17 kDa
GSH:
Glutathione
GSTZ1-1:
Glutathion transferase zeta
H:
h:
Hours
H2O:
Water
H2O2:
Hydrogen peroxide
HCC:
Hepatocellular carcinoma
x
LIST OF ABBREVIATIONS
HEPES:
N-2-Hydroxyethylpiperazine-N'-2-Ethanesulfonic Acid
HGD:
homogentisate-1,2-dioxygenase
HMPA:
High melting point agarose
HPD:
4-hydroxyphenylpyruvate dioxygenase
HPRT1:
Hypoxanthine phosphoribosyltransferase 1
HR:
Homologous repair
HRM:
High resolution melt
HT1:
Hereditary tyrosinemia 1
I:
i.e.:
id est (that is)
K:
KCl:
Potassiumchloride
(KH2P)O4:
Potassiumdihydrogen orthophosphate
L:
LMPA:
Low melting point agarose
M:
3-meA:
3-Methyladenine
7-meG:
7-Methylguanine
M:
Molar
MA:
Maleylacetone
MAA:
Maleylacetoacetate
MEM:
Minimum essential medium Eagle
mg:
Milligram
min:
Minutes
miRNA:
Micro RNA
ml:
Millilitre
MLH:
MutL homolog
mM:
Millimolar
MMLV:
Moloney Murine Leukemia Virus Reverse Transcriptase
MMR:
Mismatch repair
mRNA:
Messenger RNA
MSH:
MutS homolog
MSI:
Microsatellite instability
MutLĮ:
MLH1•PMS2 heterodimer
MutSĮ:
MSH2•MSH6 heterodimer
xi
LIST OF ABBREVIATIONS
N:
N.A.
Not available
NaCl:
Sodium chloride
NaHCO3:
Sodium bicarbonate
NaOH:
Sodium hydroxide
NCI:
National Cancer Institute
N.D.:
Not done
NER:
Nucleotide excision repair
ng:
Nanogram
NHEJ:
Non-homologous end joining
NTBC :
2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione
O:
OH:
Hydroxy radical
8-oxoG:
8-Oxoguanine
P:
PAGE:
Polyacrylamide gel electrophoresis
PBS:
Phosphate buffered saline
PCR:
Polymerase chain reaction
pH:
Potential of Hydrogen
pHPAA:
p-Hydroxyphenylacetic acid
pHPLA:
p-Hydroxyphenyllactic acid
pHPPA :
p-Hydroxyphenylpyruvic acid
PIN:
Point mutation instability
PKU:
Phenylketonuria
PMS2:
postmeiotic segregation increased 2
R:
Ref:
Reference
Rev:
Reverse
RFLP:
Restriction fragment length polymorphism
RNA:
Ribonucleic acid
RNAi:
RNA interference
RNAPII:
RNA polymerase II
ROS:
Reactive oxygen species
RPA:
Replication protein
RPM:
Revolutions per minute
RQ:
Relative quantification
rRNA:
Ribosomal RNA
RT-PCR:
Reverse transcription polymerase chain reaction
xii
LIST OF ABBREVIATIONS
S:
SA:
Succinylacetone
SAA:
Succinylacetoacetic acid
SCGE:
Single cell gel electrophoresis
sec:
Seconds
SFM:
Serum free medium
shRNA:
Short hairpin RNA
siRNA:
Small interfering RNA
SOC:
Super optimal broth with catabolite repression
SSB:
Single strand break
T:
T:
Thymine
T c:
Critical melting temperature
TCR:
Transcription coupled repair
Tet:
Tetracycline
TFIIH:
Transcription factor II H
T m:
Melting temperature
Tris-HCl:
2-Amino-2-(hydroxymethyl)-1,3-propandiol-hydrochloride
tTS:
Tetracycline-controlled transcriptional suppressor
U:
U:
Uracil
U:
Units
UV:
Ultra violet
X:
XPA:
Xeroderma pigmentosum, complementation group A
XPC:
Xeroderma pigmentosum, complementation group C
XPF:
Xeroderma pigmentosum, complementation group F
XPG:
Xeroderma pigmentosum, complementation group G
XRCC1:
X-ray repair complementing defective repair in Chinese hamster cells 1
xiii
List of figures
Figure 2-1. Tyrosine catabolism. ...........................................................................................................7
Figure 2-2. Schematic depiction of positions of mutations occurring in the fah gene. ..........................9
®
Figure 3-1. Efficiency of transfection with FuGENE . . ........................................................................28
Figure 3-2. Expression of fah by HepG2 cells after transient transfection with each of the shRNA
constructs. .........................................................................................................................29
Figure 5-1. Comet assay results of recuperation time allowed after harvesting of HepG2 cells. ...... 45
Figure 5-2. Expression of hOGG1 by HepG2 cells after exposure to a combination of 50 µM SA and
100 µM pHPPA. .............................................................................................................. 48
Figure 5-3. Expression of ERCC1 by Hepg2 cells after exposure to a combination of 50 µM SA and
100 µM pHPPA. .............................................................................................................. 49
Figure 5-4. Expression of hOGG1 and ERCC1 by HepG2 cells after exposure to SA or pHPPA
seperately. ........................................................................................................................ 50
Figure 5-5. Expression of fah, hOGG1 and ERCC1 by control and patient primary lymphocytes. .... 54
Figure 5-7. Electrophoretograms of the different microsatellite markers. .......................................... 56
Figure 5-8. Microsatellite analysis of the five microsatellite markers as recommended by the
Bethesda panel. ............................................................................................................... 57
Figure 5-9. DNA methylation in control and HT1 patients. . ................................................................ 59
Figure 5-10. Position of Lesch-Nyhan disease causing mutations on the human hprt1 gene. .......... 62
Figure 5-11. Fluorescence-normalised melting data from HRM of human fah amplicons. ................ 62
Figure 5-12. Multiple aligned fah sequencing results. ........................................................................ 63
Figure 5-13. HRM results of the fah fragment, amplified by COLD-PCR. .......................................... 64
Figure 5-14. Fluorescense-normalised melting data from HRM of human hprt1 amplicons. ............. 65
Figure 5-15. Multiple aligned human hprt1 sequencing results. ......................................................... 65
Figure 5-16. HRM results from HPP4 fragment of mouse hprt1. ....................................................... 66
Figure 5-17. Multiple aligned mouse hprt1 sequencing results after conventional PCR. .................. 67
Figure 5-18. Multiple aligned mouse hprt1 sequencing results after COLD-PCR. ............................. 67
Figure A-1. RNAi-Ready pSIREN-RetroQ-TetP vector map. ........................................................... 108
Figure A-2. ptTS-Neo vector map. ................................................................................................... 108
Figure A-3. pEGFP-N1 vector map. ................................................................................................. 109
Figure A-4. pEZSeq vector map. ...................................................................................................... 109
Figure C-1. HRM results from HPP1 fragment of mouse hprt1. ...................................................... 111
Figure C-2. HRM results from HPP5 fragment of mouse hprt1. ...................................................... 111
xiv
List of tables
Table 2-1. Summary of the inborn errors of metabolism of the tyrosine degradation pathway. .......... 6
Table 2-2. Summary of the observed mutation reversions and their effects as seen in HT1. ........... 12
Table 3-1. Sequence information of the four shRNAs designed for fah knock-down. ....................... 24
Table 3-2. Percentage knock-down of fah achieved in HepG2tTS cell lines 10 and 12. ................... 31
Table 3-3. Percentage knock-down of fah by different colonies after incubation in different growth
media. .............................................................................................................................. 32
Table 5-1. Motivation for use of the specific genes of interest. .......................................................... 47
Table 5-2. Information on microsatellite markers used for MSI testing in mouse DNA. ..................... 54
Table 5-3. Microsatellite repeat motifs. .............................................................................................. 55
xv