The incidence of Squamous Cell Carcinoma is growing in certain populations to the extent that it is now the most common skin lesion in young men and women in high ultraviolet exposure regions such as Queensland. In terms of incidence up to 45% of the Australian population over 40 years of age is thought to possess the precancerous Solar Keratosis lesion and with a small but significant chance of progression into SCC, understanding the genetic events that play a role in this process is essential. The major aims of this study were to analyse whole blood derived samples for DNA aberrations in genes associated with tumour development and cellular maintenance, with the ultimate aim of identifying genes associated with non-melanoma skin cancer development. This study had an explicit emphasis on the mitochondrial genome and nuclear genes that encode for subunits in the mitochondrial regulated energy transducing oxidative phosphorylation pathways. More specifically the first aim of this project was to analyse the NDUFA8, PTCH, NDUFAS, SMOH, SDHD, MMPI2, NDUFV1, EMSI, COXVIIc, and RASAI genes via non-specific fluorophoric Real-Time PCR for genetic aberrations in an affected Solar Keratosis and control cohort. The second aim was to analyse two specific genes, SDHD and MMPI2, for copy number aberrations via Dual-Labelled Probe Real-Time PCR in the same affected Solar Keratosis and control cohort. The third aim was to analyse Mitochondrial DNA Depletion syndrome (MDS) in a chemically exposed RAAF personnel cohort via Dual-Labelled Probe Real-Time PCR. The significance of these studies is in their contribution to the knowledge of the genetic pathways that are malformed in the progression and development of the pre-cancerous skin lesion Solar Keratosis. Furthermore, it would determine whether the genes analysed in this study exist in greater prevalence in the affected Solar Keratosis population compared to the control cohort. With regard to the MDS component, identifying the presence of this disease in these individuals was initially undertaken as part of a study to provide evidence in compensation claims. The diagnosis may assist in their medical therapy, insofar as some of them were now suffering from liver malfunctions and atypical male breast cancer. Another application of this effective and low cost method of diagnosing MDS is in populations with high HTV incidences. This is due to the fact that the most common drug used to treat this disease can give rise to the expression of MDS, thus further complicating the health status of HIV infected individuals. The analysis of this research was accomplished via the Real-Time PCR technique, with a non-specific fluorophore component in addition to specific Dual-Labelled Probe components, to ascertain the general nature of any aberration identified in the sample cohort. This project also employed additional methods of analysis such as DHPLC and DNA sequencing to assist in determining the veracity of its aims, particularly in terms of the precise detection of genetic aberrations via Real-Time PCR. Patients exhibiting male breast cancer and liver malftinctions were also analysed via Dual-Labelled Probe RealTime PCR to ascertain the presence of Mitochondrial DNA Depletion syndrome, a disorder characterised by lactic acidosis, liver failure, seizures, and congestive heart failure. Determining the presence of this syndrome in these patients would assist in their medical treatment, and contribute to the analytical methods available to diagnose this syndrome, which is known to occur in HIV sufferers due to the nucleoside drugs used to combat the disease. Real-Time PCR can adequately gauge the integrity of a genetic area in terms of amplicon malformities (non-specific-fluorophoric) and DNA copy number aberrations (Dual-Labelled Probe) via fluorophore signal differentials compared to wild-type samples and housekeeper profiles. The results of the first component of this project, namely the analysis of five gene pairs by non-specific fluorophoric Real-Time PCR, highlighted that a significantly higher incidence of putative aberrants is evident in the affected population when compared to the control cohort. The genes analysed were NDUFA8, PTCH, NDUFA5, SMOH, SDHD, MMP 12, NDUFVI, EMS 1, COXVIIc, and RASA 1. These ten genes were subdivided into five pairs; one of the pair being a gene associated with the development of a non-melanotic skin cancer (NMSC), the other a gene encoding for a subunit of the Electron Transport Chain (ETC). Each of these pairs exists in close proximity to one another on a particular chromosomal locale. Differences were highlighted in the single gene triplicate run population. The ETC genes (NDUFA8, NDUFA5, SDHD, NIDUFVI, COXVIIc) exhibited 10 / 720 (1.37%) as being putative mutants in the control population, compared to 117 / 675 (17.3%) for the affected population (p value less than 0.0001). The NMSC gene analysis (PTCH, SMOH, MMPI2, EMSI, RASA1) produced a 16 / 720 (2.22%) ratio for the control population, with the affected population having an incidence of 97 / 675 (14.4 %) for putative mutants (p value less than 0.0001). The observance of putative aberrants in the NDUFVI (p less than 0.018), EMS1 (p less than 0.003), COXVTIc (p less than 0.001), and RASA I (p less than 0.009) genes in the affected Solar Keratosis (SK) population was significantly higher than that observed in the control population. The majority of aberrations detected via the non-specific fluorophoric Real-Time PCR technique were small nucleotide base insertions and deletions. The analysis of the SK affected and control cohort via Real-Time PCR proved a cost-effective and reliable method in identifying the presence of DNA aberrations such as non-instructional sites. The results of the second component extended the findings of the non-specific fluorophoric analysis. The SDHD and MMPI 2 genes were analysed for copy number aberrations via Dual-Labelled Probe Real-Time PCR for genetic aberrations the same affected and control Solar Keratosis cohort. It was found that 12 of 279 samples had identifiable copy-number aberrations in either the SDHD or MMPI2 gene (this means that a genetic section of either of these two genes is aberrantly amplified or deleted), with five of the samples exhibiting aberrations in both genes. The MMPI2 gene also had nine samples identified as possessing an intronic heterozygous base-pair substitution anomaly via DNA sequencing. The NDUFA8 gene had 12 samples identified as anomalous via the DHPLC technique, 11 of which were identified via non-specific fluorophoric Real-Time PCR, with the analysis performed to verify the accuracy of the Real-Time technique in identifying DNA aberrations. This study identified DNA aberrations in an affected Solar Keratosis and control cohort and ascertained several particular genomic abnomialities in the SDHD, MMPI2 and NDUFA8 genes, with an emphasis on copy-number aberrations and amplicon abnormalities. In the third component of this study, namely the analysis of Mitochondrial DNA Depletion syndrome (MDS) in a jet-fuel exposed RAAF personnel cohort via Dual-Labelled Probe Real-Time PCR, the results indicated that four of the seven patients were expressing MDS. Of the four patients who exhibited a reduction in mitochondrial copy-number the average decrease was of a four-fold level, or approximately a depletion of mitochondrial copies from 200 plus to ~ 54 (74 % reduction in MtDNA). The patients who contributed DNA for investigation into the presence of MDS were suffering from liver malfunction and atypical male breast cancer. The Dual-Labelled Probe technique proved a reliable and cost effective method in identifying the presence of MDS in these patients, with the DNA extracted from fresh white blood cells that had been isolated using the Ficoll-Hypaque method. The importance of this is that accurate levels of Mitochondrial DNA copy numbers can be ascertained in white blood cells as it removes the presence of platelets, which also contain mitochondria but no nucleus. The analysis of ETC and NMSC associated genes in addition to mitochondrial copy number integrity means that this study investigated two aspects of the carcinogenetic pathway i.e. abnormal energy regulation and the regulation of micromolecular and macromolecular cellular homeostatic mechanisms. The mechanism of programmed cell death or apoptosis is regulated by the mitochondria and the ability of a genetically damaged cell to evade the apoptotic process is directly linked to a cell becoming cancerous. It is only after the evasion of apoptosis and the replication of the damaged cells' DNA into daughter cells that neoplastic events can occur. Thus, this study contributed to the understanding of how neo-plastic lesions may develop and progress into invasive tumours. It additionally assisted in proving the effectiveness of the RealTime PCR technique in detecting DNA aberrations and mitochondrial copy number anomalies.

Abstract Mitochondrial DNA depletion and deletions are related to mutations in the nuclear genes responsible for replication and maintenance of mitochondrial DNA (mtDNA). The POLG1 gene encodes the enzyme responsible for replication of mtDNA. A particular feature of the POLG1 mutations is an increased risk of acute liver failure (ALF) upon exposure to sodium valproate (VPA), but the pathomechanism is not resolved. The present work studies the molecular genetic aetiology and clinical phenotypes associated with mtDNA depletion and deletion. Another objective was an investigation of clinical phenotypes in POLG1 mutations and disentangling the pathomechanism of VPA-induced ALF in POLG1 mutations. Mitochondrial toxicity of VPA was examined using HepG2 cells as an experimental in vitro model. In this work, mtDNA depletion was associated with severe neonatal-onset encephalopathy. Furthermore, mtDNA depletion was found in muscle dystrophy as a secondary finding to muscle degradation. Multiple mitochondrial DNA deletions were found in two patients with Kearns-Sayre syndrome suggesting a genetic origin of the disease. POLG1 p.R722H mutation has been previously reported as a neutral polymorphism, but we found evidence suggesting that POLG1 p.R722H could be a pathogenic mutation in a homozygous or compound heterozygous state. We identified retrospectively five patients, who required liver transplant after VPA-induced ALF. All five patients harboured POLG1 mutations supporting the evidence of POLG1 mutations as a risk factor for VPA-induced ALF. Previously, patients with POLG1 mutations have been considered unsuitable for liver transplantation, but we found that homozygous POLG1 mutations and adolescent or adult-onset disease predicted a good outcome following liver transplantation. In vitro studies on HepG2 cells showed that VPA disturbs mitochondrial respiration. Our results expand the phenotypes and molecular genetic features in mitochondrial DNA depletion and deletion syndromes. We found evidence that POLG1 mutations are not a contraindication for liver transplantation; rather, mutation status and age at onset affect survival. This finding should be taken in consideration in the treatment of VPA-induced ALF. Furthermore, our findings indicate that sodium valproate is toxic to mitochondria and should be avoided in patients with mitochondrial disease. / Tiivistelmä Mitokondrion DNA:n (mtDNA) kahdentumisesta ja ylläpidosta vastaavien tuman geenien mutaatiot voivat johtaa mtDNA:n määrän vähenemiseen (depleetioon) ja katkoksiin (deleetioihin). MtDNA:n kahdentumisesta vastaavaa entsyymiä koodaa tuman POLG1-geeni. POLG1-mutaatioihin liittyy kohonnut riski sairastua natriumvalproaatin (VPA) aiheuttamaan akuuttiin maksavaurioon. Tutkimuksen tavoitteena oli tutkia mtDNA:n depleetion ja deleetioiden molekyyligeneettistä etiologiaa ja kliinisiä taudinkuvia. Tutkimuksessa selvitettiin myös POLG1-mutaatioihin liittyviä taudinkuvia ja POLG1-mutaatioihin liittyvän akuutin maksavaurion patomekanismia. VPA:n vaikutusta mitokondrioiden toimintaan tutkittiin in vitro HepG2-solumallissa. Tutkimuksessa todettiin mtDNA:n depleetion liittyvän vaikeaan varhain alkavaan aivosairauteen. Depleetio todettiin myös sekundaarisena merosiini-negatiivisessa lihasdystrofiassa. Kahdella Kearns-Sayren syndroomaa sairastavalla potilaalla todettiin multippelit mtDNA:n deleetiot, mikä viittaa syndrooman geneettisen alkuperään. POLG1 p.R722H-mutaatiota on aiemmin pidetty neutraalina polymorfiana, mutta tutkimuksen tulokset viittasivat siihen, että homotsygoottisena tai yhdistelmäheterotsygoottisena mutaatio on tautia aiheuttava. Helsingin yliopistollisen sairaalan elinsiirtorekisteristä tunnistettiin retrospektiivisesti viisi potilasta, jotka olivat saaneet maksansiirteen VPA:n aiheuttaman maksavaurion vuoksi. Kaikilla viidellä potilaalla todettiin POLG1-geenin mutaatio, mikä vahvistaa käsitystä geenin yhteydestä VPA:n aiheuttamaan maksavaurioon. POLG1-mutaatioita on pidetty vasta-aiheena maksansiirrolle, mutta tutkimuksessa todettiin homotsygoottisena esiintyvän POLG1-mutaation ja nuoruusiällä tai varhaisella aikuisiällä alkaneen taudin liittyvän parempaan maksansiirron jälkeiseen ennusteeseen. HepG2-solumallilla tehdyt tutkimukset osoittivat VPA:n haittaavan mitokondrioiden solyhengitystä. Tutkimuksen tulokset tuovat lisätietoa mtDNA:n depleetioon ja deleetioihin liittyvistä taudinkuvista ja molekyyligeneettisestä taustasta. POLG1-mutaatiot eivät ole ehdoton vasta-aihe maksansiirrolle; potilaan geneettinen status ja ikä taudin alkamishetkellä vaikuttavat ennusteeseen, mikä tulisi huomioida potilaiden hoidossa. Tulokset myös osoittivat VPA:n olevan mitokondriotoksinen lääke, jonka käyttöä tulisi välttää mitokondriotautipotilaiden hoidossa.