Tesis sobre el tema "Mitochondrial DNA depletion syndrome"

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.

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.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>School of Biomolecular and Biomedical Sciences<br>Full Text

Mitochondrial DNA depletion syndromes (MDS) are a group of rare autosomal recessive disorders with early onset and no cure available. MDS are caused by mutations in several nuclear genes, involved in mitochondrial DNA (mtDNA) maintenance, characterized by a strong reduction of mtDNA copy number in affected tissues and severe defects in mitochondrial functionality. Mutations in MPV17, a nuclear gene encoding a mitochondrial inner membrane protein, have been specifically associated with hepatocerebral forms of MDS. However, MPV17 protein function is still unclear, although it has been suggested a primary role in mtDNA maintenance. Zebrafish represents a model to clarify this biological question: a mpv17 null mutant (roy orbison) shows a 19 bp deletion resulting in aberrant splicing between the exons 2 and 3 of mpv17 gene and lacks the guanine-based reflective skin cells named iridophores. In our work, we have characterized in details the mitochondrial phenotype of roy larvae and found early severe ultrastructural alterations in liver mitochondria; we could also observe a significant impairment of respiratory chain complexes leading to mitochondrial quality control activation. Our results provide evidences for Mpv17 being really essential in mitochondrial cristae maintenance and OXPHOS functionality, while its effect on mtDNA maintenance seems to be consequential, considering that mtDNA depletion only appeared at later stages of development. Moreover, taking into account that in roy orbison it has been previously postulated a role for Mpv17 in purines availability, and that embryos blocked in their pyrimidine synthesis resemble roy phenotype, we investigated the two alternatives by administrating purine and pyrimidine precursors to homozygous mutant embryos. Interestingly, orotic acid (OA) administration ameliorated roy phenotype, hence linking the loss of Mpv17 to pyrimidine de novo synthesis. In particular, the treatment with OA, currently used as food supplement, significantly increased not only iridophores number but also mtDNA content of mpv17 null mutants, thus opening up a new simple therapeutic approach for MPV17-related MDS.<br>Le sindromi da deplezione del DNA mitocondriale (MDS) sono un gruppo di malattie rare a carattere autosomico recessivo con esordio precoce e prognosi infausta. Le MDS sono causate da mutazioni a carico di diversi geni nucleari, coinvolti nel mantenimento del DNA mitocondriale (mtDNA), e caratterizzate da una forte riduzione del numero di copie del mtDNA nei tessuti interessati, nonchè da gravi difetti nella funzionalità mitocondriale. Le mutazioni a carico di MPV17, un gene nucleare che codifica per una proteina di membrana mitocondriale interna, sono state specificatamente associate a forme epatocerebrali di MDS. Tuttavia, la funzione della proteina MPV17 non è nota, sebbene sia stato ipotizzato un ruolo primario nel mantenimento del mtDNA. Zebrafish rappresenta un modello per rispondere a questa domanda biologica: un mutante nullo per il gene mpv17, detto roy orbison (roy), presenta una delezione di 19 bp che causa uno splicing aberrante tra gli esoni 2 e 3 del gene e, di conseguenza, l’assenza della proteina codificata. Dal punto di vista fenotipico, il mutante roy mostra un difetto di pigmentazione, caratterizzato dall’assenza di cellule, chiamate iridofori, le quali conferiscono alla pelle, grazie al loro contenuto di cristalli di guanina, la sua proprietà riflettente. Il presente lavoro di tesi ha avuto come obiettivo principale la caratterizzazione dettagliata del fenotipo mitocondriale delle larve mutanti per il gene mpv17, nelle quali abbiamo rilevato, già a stadi precoci, importanti alterazioni dell’ultrastruttura mitocondriale nel fegato e una significativa perdita della funzionalità dei complessi della catena respiratoria (OXPHOS). I nostri risultati suggeriscono, pertanto, una funzione essenziale del gene mpv17 nel mantenimento delle creste mitocondriali e della piena attività dell’OXPHOS, mentre il suo effetto sulla stabilità del mtDNA, ipotizzato in letteratura, sembra essere consequenziale, considerando che la deplezione del mtDNA è rilevabile solo in fasi tardive dello sviluppo. Inoltre, considerando che nei mutanti roy orbison era stata precedentemente ipotizzata una funzione di Mpv17 nel metabolismo delle purine e che, inoltre, l’inibizione della sintesi di pirimidine negli embrioni wild-type causa un fenotipo assimilabile a quello dei roy, sono stati somministrati precursori purinici e pirimidinici agli embrioni omozigoti mutanti, al fine di osservare o meno un miglioramento del fenotipo. È interessante notare che la somministrazione di acido orotico (OA), prodotto dell’enzima mitocondriale Diidroorotato deidrogenasi (DHODH) e precursore delle pirimidine, ha migliorato il fenotipo roy, collegando quindi la perdita di Mpv17 alla sintesi de novo dei nucleotidi. In particolare, il trattamento con OA, attualmente usato come integratore alimentare, ha aumentato significativamente non solo il numero di iridofori ma anche il contenuto di mtDNA nei mutanti nulli di mpv17, aprendo così nuove prospettive nel trattamento delle malattie da deplezione del DNA mitocondriale causate da mutazioni a carico di MPV17.

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<br>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

Type 2 diabetes is an age-related condition and is characterised by a progressive decline in insulin secretion. Mitochondria play a key role in energy generation for insulin secretion. We previously reported an age-related decline in mitochondrial DNA (mtDNA) copy number in isolated human islets. TFAM, mtDNA Transcription Factor A, regulates mtDNA transcription and mtDNA copy number. Aims: We aimed to replicate the percentage decrease in mtDNA copy number that we observed with ageing in human islets, and to explore whether this affected mitochondrial function and insulin secretion. Methods: Two independent models of mtDNA depletion were created. The first model knocked down TFAM gene expression using siRNA technology. The second model subjected cells to didanosine, a nucleoside analogue of adenosine with a high affinity to POLG, a mtDNA polymerase. Results: Both models produced comparable levels of mtDNA depletion. Upon investigating the effects of partial mtDNA depletion on mitochondrial function, we found that both mtDNA depletion models displayed reduced mtDNA gene transcription and translation. However, neither model of mtDNA depletion affected ATP content or mitochondrial membrane potential. Glucose-stimulated insulin secretion was decreased following mtDNA depletion in the TFAM knock down cells which was rescued following treatment with the insulin secretagogue glibenclamide. Conversely, didanosine-induced mtDNA depleted cells showed increased insulin secretion. Conclusions: Both models generated a similar degree of mtDNA depletion, which was comparable to the percentage decrease seen in human islets with ageing. Both models were seen to impair mitochondrial function, but with opposing effects on insulin secretion. The TFAM model findings are in line with previous studies of severe mtDNA depletion, suggesting that the increase in insulin secretion seen with didanosine is due to drug off target effects. Strategies to slow islet mtDNA depletion in man could help to preserve insulin secretion and delay the development of Type 2 diabetes.

Introduction. Down Syndrome (DS) is the most known autosomal trisomy, due to the presence in three copies of chromosome 21. Many studies were designed to identify phenotypic and clinical consequences related to the triple gene dosage. However, the general conclusion is a senescent phenotype; in particular, the most features of physiological aging, such as skin and hair changes, vision and hearing impairments, thyroid dysfunction, Alzheimer-like dementia, congenital heart defects, gastrointestinal malformations, immune system changes, appear in DS earlier than in normal age-matched subjects. The only established risk factor for the DS is advanced maternal age, responsible for changes in the meiosis of oocytes, in particular the meiotic nondisjunction of chromosome 21. In this process mitochondria play an important role since mitochondrial dysfunction, due to a variety of extrinsic and intrinsic influences, can profoundly influence the level of ATP generation in oocytes, required for a correct chromosomal segregation. Aim. The aim of this study is to investigate an integrated set of molecular genetic parameters (sequencing of complete mtDNA, heteroplasmy of the mtDNA control region, genotypes of APOE gene) in order to identify a possible association with the early neurocognitive decline observed in DS. Results. MtDNA point mutations do not accumulate with age in our study sample and do not correlate with early neurocognitive decline of DS subjects. It seems that D-loop heteroplasmy is largely not inherited and tends to accumulate somatically. Furthermore, in our study sample no association of cognitive impairment and ApoE genotype is found. Conclusions. Overall, our data cast some doubts on the involvement of these mutations in the decline of cognitive functions observed in DS.

Introduction. Down Syndrome (DS) is the most known autosomal trisomy, due to the presence in three copies of chromosome 21. Many studies were designed to identify phenotypic and clinical consequences related to the triple gene dosage. However, the general conclusion is a senescent phenotype; in particular, the most features of physiological aging, such as skin and hair changes, vision and hearing impairments, thyroid dysfunction, Alzheimer-like dementia, congenital heart defects, gastrointestinal malformations, immune system changes, appear in DS earlier than in normal age-matched subjects. The only established risk factor for the DS is advanced maternal age, responsible for changes in the meiosis of oocytes, in particular the meiotic nondisjunction of chromosome 21. In this process mitochondria play an important role since mitochondrial dysfunction, due to a variety of extrinsic and intrinsic influences, can profoundly influence the level of ATP generation in oocytes, required for a correct chromosomal segregation. Aim. The aim of this study is to investigate an integrated set of molecular genetic parameters (sequencing of complete mtDNA, heteroplasmy of the mtDNA control region, genotypes of APOE gene) in order to identify a possible association with the early neurocognitive decline observed in DS. Results. MtDNA point mutations do not accumulate with age in our study sample and do not correlate with early neurocognitive decline of DS subjects. It seems that D-loop heteroplasmy is largely not inherited and tends to accumulate somatically. Furthermore, in our study sample no association of cognitive impairment and ApoE genotype is found. Conclusions. Overall, our data cast some doubts on the involvement of these mutations in the decline of cognitive functions observed in DS.

Background: Nucleoside reverse transcriptase inhibitors (NRTIs) interfere with mitochondrial DNA polymerase gamma causing significant toxic effects, including fatal lactic acidosis. Little is known about mitochondrial DNA (mtDNA) in human immunodeficiency virus (HIV) infected children who face a lifetime exposure to these agents. We performed a cross sectional observation of mtDNA levels in whole blood in a pediatric population to ascertain the relationship between mtDNA, NRTI regimens and parameters of HIV-infection severity. Methods: Whole blood mt:nDNA ratios were determined by real-time PCR in three groups: 27 presumed HIV-negative, 89 HIV-infected, NRTI-treated and 62 HIV-infected treatment-naive children. Multivariate analysis was used to identify variables independently associated with mtDNA depletion. Results: Mean mt:nDNA ratios were lower (P < 0.001) at 77% of control in the HIVinfected antiretroviral treatment (ART) Naïve group and 73% of control in the ART group, but not different between the two HIV-infected groups. Mt:nDNA ratios were negatively associated with age (P = 0.029), HIV status (P < 0.0001) and Log10 of the HIV-1 viral load (P = 0.035) and positively associated with CD4 % (p = 0.032). A 6 stavudine vs zidovudine based regimen was associated with lower but not significant levels of mtDNA (P = 0.1). Conclusions: Depletion of whole blood mtDNA in children is associated independently with HIV-infection and markers of HIV infection severity, and does not improve with either stavudine or zidovudine based ART despite virological control, suggesting that these agents also deplete mtDNA.

Abstract This study describes clinical and biochemical consequences of the 3243A→G mutation in the tRNALeu(UUR) gene of the mitochondrial DNA. Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS syndrome) is usually caused by this mutation. Demyelinating polyneuropathy was observed as a novel feature in a patient with the mutation. Based on electrodiagnostic examination the polyneuropathy was defined as being of the demyelinating, mixed (motor more than sensory) type. In a 1 year follow-up an approximately 7% reduction in both the motor and sensory nerve conduction velocities were observed. The effect and mechanism of action of nicotinamide treatment in a MELAS patient with the 3243A→G was studied. The blood NAD concentration increased linearly, being 24-fold elevated at 6 weeks of treatment. Blood lactate and pyruvate concentration decreased by 50% within three days and 24 h urine lactate content within 2 weeks. A clinical improvement together with a decrease in the lesion volume in magnetic resonance imaging within the first month were observed. Alleviation of the lactate accumulation during the nicotinamide treatment suggested that an increase in the cellular NAD+NADH concentration led to enhancement of the oxidation of reducing equivalents, suggesting that complex I of respiratory chain operates at non-saturating substrate concentration. Myoblasts cultured from patients carrying the 3243A→G mutation and from controls were used to measure ATP, ADP, catalase and superoxide dismutase activity, population growth, apoptotic cell death and the morphology of cytoskeletal components. ATP and ADP concentrations were decreased, suggesting a decrease in the adenylate pool. The superoxide dismutase and catalase activities were higher than in control cells, suggesting an increased production of reactive oxygen species due to respiratory chain dysfunction. No increase in apoptotic cell death was observed in proliferating myoblasts, but randomization of vimentin filament direction and length was observed and decreased population growth was associated with the mutation. The results show that the 3243A→G mutation leads to numerous secondary pathophysiological events. Based on the literature and the results of this study, similarities were found between the pathophysiology of 3243A→G mutation and other neurodegenerative diseases and aging.

Plant mitochondrial genomes are large and complex, and the mechanisms for maintaining mitochondrial DNA (mtDNA) remain unclear. Arabidopsis thaliana has two DNA polymerase genes, polIA and polIB, that have been shown to be dual localized to mitochondria and chloroplasts but are unequally expressed within primary plant tissues involved in cell division or cell expansion. PolIB expression is observed at higher levels in both shoot and root apexes, suggesting a possible role in organelle DNA replication in rapidly dividing or expanding cells. It is proposed that both polIA and polIB are required for mtDNA replication under wild type conditions. An Arabidopsis T-DNA polIB mutant has a 30% reduction in mtDNA levels but also a 70% induction in polIA gene expression. The polIB mutant shows an increase relative to wild type plants in the number of mitochondria that are significantly smaller in relative size, observed within hypocotyl epidermis cells that have a reduced rate of cell expansion. These mutants exhibit a significant increase in gene expression for components of mitorespiration and photosynthesis, and there is evidence for an increase in both light to dark (transitional) and light respiration levels. There is not a significant difference in dark adjusted total respiration between mutant and wild type plants. Chloroplast numbers are not significantly different in isolated mesophyll protoplasts, but mesophyll cells from the mutant are significantly smaller than wild type. PolIB mutants exhibit a three-day delay in chloroplast development but after 7dpi (days post-imbibition) there is no difference in relative plastid DNA levels between the mutant and wild type. Overall, the polIB mutant exhibits an adjustment in cell homeostasis, which enables the maintenance of functional mitochondria but at the cost of normal cell expansion rates.

Mitochondria are membrane bound organelles important for energy production in respiring cells through the process of oxidative phosphorylation. They have their own multi-copied mitochondrial DNA (mtDNA) genome separate from that in the nucleus that is needed for mitochondria to function properly and can exist in both wild type and mutant forms in the same cell. The integrity of the mtDNA is therefore of vital importance for the survival of the organism and as such understanding the mechanisms of mtDNA maintenance is relevant to human health and disease. This study employs a Southern blotting and non-radioactive probe method to examine various aspects of mtDNA maintenance. Restriction endonuclease mapping utilizing mtDNA-specific and nuclear DNA-specific digoxigenin (DIG)-labeled probes was performed to show that the synthesized probes are indeed specific for their target sequences. The DIG-labeled probes were used to quantitate mtDNA content from different DNA isolation methods. Whole-cell DNA extraction was found to yield higher levels of mtDNA compared to a commercially available spin-column kit. Next, Southern blots were used to analyze mtDNA copy number as well as mtDNA depletion in the hepatocarcinoma-derived cell line HepaRG following exposure to the nucleoside reverse transcriptase inhibitor 2’,3’-dideoxycytidine (ddC), a known mitochondrial toxicant. In comparison to proliferative HepaRG differentiated HepaRG contained about 2-fold more mtDNA. Relative to untreated control cells, proliferating HepaRG exposed to ddC had greater than a 96% reduction in mtDNA and had decreased cellular viability. Differentiated HepaRG cell viability was not affected after 13 days of ddC treatment; however, significant mtDNA depletion was observed. We estimate that differentiated HepaRG mtDNA depletion occurs quickly at about 20 molecules per hour.

Abstract Childhood acute lymphoblastic leukemia (ALL) is the most common cancer in children. The overall survival rate has reached to 90%. However, ALL still presents a significant disease burden and is a major cause for deaths in children. Recently, both inherited germline variants related to ALL susceptibility and somatic genetic variants forming novel subgroups of ALL have been discovered. In this thesis two families with familial ALL were studied. Constitutional heterozygous microdeletion at chromosome 7p12.1p13, including IKZF1, was discovered in the first family with intellectual impairment, overgrowth, and susceptibility to childhood ALL. In the second family, constitutional chromosome translocation was revealed in two individuals with childhood ALL and, subsequently, in seven unaffected family members. The balanced reciprocal translocation t(12;14)(p13.2;q23.1) resulted in breakpoints on two genes; ETV6 on chromosome 12 and RTN1 on chromosome 14. Only a few familial and sporadic ALL cases with germline variants in either IKZF1 or ETV6 have been published, thus supporting the significant role of these constitutional variants in childhood ALL predisposition. Inherited bone marrow failure syndromes (IBMFS) may predispose to childhood leukemia, including ALL. Two unrelated patients were diagnosed with bone marrow failure without the symptoms of classical IBMFS. Neither patient had any signs of developmental delay or congenital anomalies. Exome sequencing revealed identical c.1457del(p.(Ile486fs)) mutation on the ERCC6L2 gene in both patients. A few patients with IBMFS and ERCC6L2 variants have been described in previous studies. Some of them also had congenital craniofacial anomalies and developmental delay that were not detected in the patients in this thesis. The ALL cohort study on genetic variation of mitochondrial DNA (mtDNA) included 36 children. Metabolic change where malignant cells uncouple energy production from oxidative phosphorylation (OXPHOS) is one of the established hallmarks of cancer. In the cohort in this study, 22% of patients harbored nonsynonymous variants on mtDNA in the protein-coding genes of OXPHOS enzyme complexes. The somatic non-neutral variants were found in patients with a poor prognosis cytogenetic marker. The results support the hypothesis that cancer cells harbor mtDNA variants that may affect the cell metabolism<br>Tiivistelmä Akuutti lymfoblastileukemia (ALL) on lasten yleisin syöpä. Vaikka nykyisin noin 90 prosenttia paranee, ALL aiheuttaa huomattavan paljon sairastavuutta ja on merkittävä lasten kuolinsyy. Vastikään on löydetty perinnöllisiä geneettisiä muutoksia, jotka altistavat lapsuusiän ALL:lle. Tutkimuksen kohteena oli kaksi perhettä, joissa vähintään kaksi lasta on sairastunut ALL:aan. Ensimmäisessä perheessä havaittiin lapsuusiän ALL:aan sairastuneilla kehityshäiriöisillä sisaruksilla äidiltä periytyvä heterotsygoottinen deleetio kromosomissa 7p12.1p13, jossa sijaitsee IKZF1-geeni. Toisessa perheessä perinnöllinen kahden kromosomin translokaatio todettiin kahdella lapsuusiän ALL:aan sairastuneella sekä seitsemällä perheenjäsenellä. Balansoitu translokaatio t(12;14)(p13.2;q23.1) aiheuttaa katkaisukohdan ETV6-geeniin kromosomissa 12 ja RTN1-geeniin kromosomissa 14. Tähän mennessä on julkaistu vain muutamia tutkimuksia potilaista, joilla on ollut perinnöllinen muutos joko IKZF1- tai ETV6-geenissä. Näillä geeneillä oletetaan olevan tärkeä merkitys perinnöllisessä alttiudessa sairastua lapsuusiän ALL:aan. Perinnölliset luuytimen toimintahäiriöt voivat altistaa leukemialle, kuten ALL:lle. Kahdella lapsella todettiin luuytimen toimintahäiriö, mutta ei muita oireita, jotka voisivat liittyä tyypillisiin perinnöllisiin luuytimen toimintahäiriöihin. Eksomisekvensoinnissa todettiin identtinen, homotsygoottinen mutaatio c.1457del(p.(Ile486fs)) ERCC6L2-geenissä. Kirjallisuuslähteiden mukaan vain muutamalla potilaalla on todettu ERCC6L2-geenin muutoksesta johtuva luuytimen toimintahäiriö. Osalla heistä on ollut synnynnäisiä kallon ja kasvojen anomalioita sekä kehityshäiriö, jollaisia tähän tutkimukseen osallistuneilla potilailla ei todettu. Potilaskohorttitutkimuksessa tutkittiin mitokondriaalisen DNA:n (mtDNA) muutoksia ALL:aan sairastuneilla lapsilla. Syöpäsolut eivät hyödynnä mitokondrion elektroninsiirtoketjua energian tuotantoon, ja tämä aineenvaihdunnan muutos on tunnustettu syövän ominaisuus. Tutkimuksessa havaittiin, että 22 prosentilla potilaista ilmeni diagnoosivaiheessa poikkeavia mtDNA:n muutoksia, jotka olivat elektroninsiirtoketjun entsyymien alayksiköitä koodaavissa geeneissä. Muutoksia todettiin useimmiten potilailla, joilla oli leukemiasoluissa huonon ennusteen geneettinen tekijä. Havaitut muutokset voivat mahdollisesti vaikuttaa leukemiasolun energia-aineenvaihduntaan

Human mitochondrial DNA (mtDNA) is located in discrete DNA-protein complexes, so called nucleoids. These structures can be easily visualized in living cells by utilizing the fluorescent stain PicoGreen®. In contrary, cells devoid of endogenous mitochondrial genomes (ρ0 cells) display no mitochondrial staining in the cytoplasm. A modified restriction enzyme can be targeted to mitochondria to cleave the mtDNA molecules in more than two fragments, thereby activating endogenous nucleases. By applying this novel enzymatic approach to generate mtDNA-depleted cells the destruction of mitochondrial nucleoids in cultured cells could be detected in a time course. It is clear from these experiments that mtDNA-depleted cells can be seen as early as 48 h post-transfection using the depletion system. To prove that mtDNA is degraded during this process, mtDNA of transfected cells was quantified by real-time PCR. A significant decline could be observed 24 h post-transfection. Combination of both results showed that mtDNA of transfected cells is completely degraded and, therefore, ρ0 cells were generated within 48 h. Thus, the application of a mitochondrially-targeted restriction endonuclease proves to be a first and fast, but essential step towards a therapy for mtDNA disorders.

Abstract NADH-ubiquinone oxidoreductase (complex I) is one of the largest enzymes in mammals. Seven (ND1-ND6 and ND4L) of its 45 subunits are encoded in mitochondrial DNA, mutations of which are usually behind mitochondrial diseases such as Leber hereditary optic neuropathy (LHON) and MELAS-syndrome. The rest of the genes are located in the nucleus. Bacterial homologs of complex I (NDH-1) consist of only 13&#8211;14 subunits, comprising the catalytic core of the enzyme. These complexes are simpler but perform a similar function. Escherichia coli NDH-1 was employed here to generate amino acid replacements at conserved sites in NuoH, NuoJ and NuoK, counterparts of ND1, ND6 and ND4L, to elucidate their role in complex I. Consequences of homologous amino acid substitutions brought about by ND1-affecting LHON/MELAS-overlap syndrome-associated m.3376G&gt;A and m.3865A&gt;G mutations and the ND6-affecting m.14498T&gt;C substitution associated with LHON were also studied to validate their pathogenicity. Effects of the site-directed mutations were evaluated on the basis of enzyme activity, inhibitor sensitivity and growth phenotype. Highly conserved glutamate-residues 36 and 72 within transmembrane helices of NuoK in positions similar to proton translocating transmembrane proteins were found essential for electron transfer to ubiquinone and growth on medium necessitating normal proton transfer by NDH-1. NuoH and NuoJ replacements at sites corresponding to targets of m.3376G&gt;A and m.14498T&gt;C decreased ubiquinone reductase activity and altered the ubiquinone binding site, while the counterpart of m.3865A&gt;G was without a major effect. Other NuoH and NuoJ mutations studied also affected the interactions of ubiquinone and inhibitors with NDH-1. The results corroborate the pathogenicity of the m.14498T&gt;C and m.3376G&gt;A mutations and demonstrate that the overlap syndrome-associated modification affects complex I in a pattern which appears to combine the effects of separate mutations responsible for LHON and MELAS. Change in ubiquinone binding affinity is a likely pathomechanism of all LHON-associated mutations. Effects of the NuoH, NuoJ and NuoK subunit substitutions also indicate that ND1 and ND6 subunits contribute to the ubiquinone-interacting site of complex I and the site is located in the vicinity of the membrane surface, while ND4L is likely involved in proton pumping activity of the enzyme<br>Tiivistelmä 45 alayksiköstä muodostuva NADH-ubikinoni oksidoreduktaasi (kompleksi I) on nisäkkäiden suurimpia entsyymejä. Sen mitokondriaalisessa DNA:ssa koodattujen alayksiköiden ND1-ND6 ja ND4L geeneihin liittyvät mutaatiot ovat yleisiä mitokondriosairauksien, kuten Leberin perinnöllisen näköhermoatrofian (LHON) ja MELAS-oireyhtymän, syitä. Bakteerien vastaava entsyymi (NDH-1) koostuu vain 13&#8211;14 alayksiköstä. Tästä huolimatta sen katalysoima reaktio on samankaltainen kuin kompleksi I:n. NDH-1:n katsotaankin edustavan entsyymin katalyyttistä ydintä. Tässä työssä tutkittiin ND1, ND6 ja ND4L alayksiköiden tehtävää kompleksi I:ssä niiden Escherichia coli bakteerissa olevien vastineiden (NuoH, NuoJ ja NuoK) kohdennetun mutageneesin avulla. Samaa lähestymistapaa käytettiin LHON/MELAS-oireyhtymässä todettujen ND1 alayksikön mutaatioiden, m.3376G&gt;A ja m.3865A&gt;G, ja LHON:ssa havaitun ND6:n m.14498T&gt;C mutaation aiheuttamien aminohappomuutosten seurauksien selvittämiseen. Tehtyjen mutaatioiden vaikutuksia arvioitiin entsyymiaktiivisuus-mittauksin ja kasvukokein. NuoK:n solukalvon läpäisevissä rakenteissa olevien kahden glutamaatti-aminohappotähteen sijainti muistuttaa protoneita kalvon läpi kuljettavissa proteiineissa todettua. NuoK:n glutamaattien havaittiinkin olevan tärkeitä elektronien ja protonien kuljetukselle kompleksi I:ssä. m.3376G&gt;A ja m.14498T&gt;C mutaatioiden aiheuttamien aminohappomuutosten vastineet NDH-1:ssä alensivat NDH-1:n elektroninsiirtoaktiivisuutta ja heikensivät ubikinonin sitoutumista, kun taas m.3865A&gt;G mutaatiolla ei ollut vaikutusta. Muut NuoH ja NuoJ alayksiköihin tehdyt aminohappovaihdokset johtivat huonontuneeseen ubikinonin ja kompleksi I:n inhibiittoreiden sitoutumiseen. Saadut tulokset vahvistavat m.3376G&gt;A ja m.14498T&gt;C mutaatioiden patogeenisyyden. Ne myös osoittavat, että LHON/MELAS-oireyhtymään liitetyn mutaation biokemiallisissa vaikutuksissa yhdistyvät sekä LHON:ssa että MELAS-oireyhtymässä todettujen mutaatioiden seuraukset. Esitetyt tulokset tukevat näkemystä siitä, että ubikinonin ja kompleksi I:n välisessä vuorovaikutuksessa tapahtuva muutos on kaikille LHON:aan liitetyille mutaatioille yhteinen vaikutusmekanismi. NuoH:n, NuoJ:n ja NuoK:n kohdennetusta mutageneesista saatujen tulosten perusteella ND1 ja ND6 alayksiköt ovat osa ubikinonin sitoutumispaikkaa entsyymikompleksissa, kun taas ND4L osallistuu protoninkuljetukseen

Trabalho final de mestrado integrado em Medicina (Bioquímica), apresentado à Faculdade de Medicina da Universidade de Coimbra<br>O défice da desoxiguanosina cinase (dGUOK), causado por mutações no gene DGUOK, leva ao desenvolvimento da forma hepatocerebral da Síndrome de depleção de DNA mitocondrial (MDS), ligadas a uma redução drástica no número de cópias do genoma mitocondrial (mtDNA), associada a elevada mortalidade e sem estratégias terapêuticas disponíveis, na criança. Apesar de se terem vindo a identificar novas mutações no gene DGUOK ao longo dos últimos anos, uma correlação genótipo-fenótipo precisa e exequível, nunca foi estabelecida. Um padrão reprodutível de associação entre o tipo e localização das mutações genéticas no gene DGUOK e a clínica apresentada pelo doente facultaria aos clínicos a capacidade de estimar, num estádio neonatal, ou mesmo pré-natal, o diagnóstico e a gravidade da doença, provendo os clínicos com ferramentas mais racionais para aconselhamento genético adequado às famílias e aos futuros pais. Procedeu-se a uma busca extensiva na base de dados de referências MEDLINE para procurar todas as mutações no gene DGUOK, publicadas até à data. Estas alterações foram analisadas quanto à sua localização no gene e o seu impacto na função e estrutura da proteína, assim como nas manifestações clínicas apresentadas pelos doentes e foi efetuado um estudo da correlação genótipo-fenótipo. Verificou-se a inexistência de uma correlação genótipo-fenótipo precisa e clara em doentes com MDS causado por mutações no gene DGUOK, revelando a existência de mecanismos mais complexos e ainda desconhecidos, que estarão subjacentes ao início e à progressão da doença<br>Deoxyguanosine kinase (coded by DGUOK) deficiency, caused by DGUOK gene mutations, leads to development of the hepatocerebral form of mitochondrial DNA depletion syndrome (MDS), linked to mitochondrial genome (mtDNA) copy number severe decline, associated to high mortality and absence of available therapeutic strategies in infants. In spite of the continuous identification of new DGUOK mutations over the years, an accurate and feasible genotype-phenotype correlation in DGUOK patients is lacking. A reproducible pattern of association between the type and localization of DGUOK gene mutation and its effect on patients´ clinic would ground physicians with the capacity to foresee, at a neonatal or even pre-natal stage, the disease diagnostics and severity, providing the clinicians, therefore, with more rationale tools for adequate genetic counselling in families and future parents. An extensive search on MEDLINE database was performed in order to search for all published DGUOK gene mutations to date. These alterations were analysed for their localization in the gene and their impact on protein structure and function and the clinical manifestations and a genotype-phenotype correlation study has been carried out. It was evident the absence of a clear and accurate genotype-phenotype correlation in patients with MDS caused by DGUOK mutations, unmasking, likely, more complex and unknown mechanisms underlying disease onset and progression

碩士<br>臺北醫學大學<br>醫學技術學系<br>93<br>Abstract The Sudden Infant Death Syndrome (SIDS) is one kind of leading cause of postneonatal infant death which also rises a difficult problem between the legal and medical systems. SIDS is defined as the sudden death of an infant less than 12 month old that remains unexplained after a complete clinical review, autopsy and death scene investigation. Despite the fact that many hypotheses have been proposed, the causes of SIDS are still uncertain. Although a number of coding region mtDNA mutations involving SIDS have been reported, the role of mtDNA variations in SIDS victims is still not known. The purpose of this study was to investigate whether the mtDNA variations or large-scale deletion exert any effect on the etiology of SIDS. Moreover, the biologic significance of the hOGG1 1245C"G polymorphism for SIDS has not yet been elucidated. The polymorphism of the hOGG1 gene was assessed by using a PCR-restriction fragement length polymorphism (RFLP) method. Seven SIDS and nineteen non-SIDS victims were enrolled. We determined the relative amount of mtDNA copy number and the occurrence of mtDNA deletion in blood, skeletal muscle and cardiac muscle from both SIDS and non-SIDS group using real-time quantitative PCR, primer-shift PCR analysis and DNA sequencing. Buccal epithelial samples from twenty age-matched live health control subjects were also examined. In this study we also explore the DNA sequence variation of D-loop hypervariable region I(HVR I) and HVR II. D-loop sequence of all subjects were sequenced and compared with the Cambridage sequence. The mean number of substitutions in HVR I and HVR II between SIDS, non-SIDS and live health control were no significant difference. Three types of mtDNA deletions were found in this study such as 4977, 5335 and 7599 bp deletion. The breakpoints of deletion were identified by sequencing method. In live health control group only one subject was found with 7599 bp deletion from buccal epithelial sample. Only one specimen was found with 4977 bp mtDNA deletion from cardiac muscle in congenital heart malformation subject. The frequencies of occurrence of 5335 and 7599 bp deletions in SIDS and non-SIDS were much higher than live health control group. There were no statistically significant difference associated with the frequencies of occurrence of 5335 bp and 7599 bp mtDNA deletions between SIDS and non-SIDS victims was found. The frequency of occurrence of 5335 and 7599 bp mtDNA deletion in blood from SIDS were four and two fold to non-SIDS, respectively. The frequency of occurrence of 5335 bp mtDNA deletion in skeletal muscle was 1.8 fold to non-SIDS. No significant correlation between the relative amount of mtDNA copy number and the frequencies of occurrence of mtDNA deletions in SIDS and non-SIDS. The allelic frequency of hOGG1 1245G was 78.6 % in SIDS, 61.1 % in non-SIDS and 45.0 % in live health control. The genotypic frequencies were no statistically significance between SIDS and live health control(p=0.062) and between non-SIDS and live health control(p=0.246). These defects in mtDNA may result in impaired production of ATP or sensitized cells to apoptosis. We proposed that mtDNA deletions in themselves do not cause SIDS but may cause energy deficiency or hypoxia in stressful situation during a vulnerable developmental stage. So the primitive results of mitochondrial DNA deletion might predispose infant to death in critical situations.

碩士<br>長榮大學<br>醫學研究所<br>97<br>Objective: The metabolic syndrome (MetS) is a group of risk factors of metabolic origin that are accompanied by increased risk for type 2 diabetes mellitus and cardiovascular disease. One of the defects in MetS and its associated diseases is excess cellular oxidative stress and mitochondrial alternation. A low copy number of mitochondrial DNA (mtDNA) has been found to be associated with enhanced risk for the development of diabetes and cardiovascular disease. Therefore, the objective of this study was to clarify the characteristics of mtDNA copy number of leukocytes in MetS. Methods: Seventy nine control subjects and 42 metabolic syndrome subjects of Taiwanese were recruited and Quantitative polymerase chain reaction (QPCR) and real time PCR were applied in present study. A student t test, multi-variate ANOVA, and Spearman''s rho correlation analysis were used for statistic analysis. Results: The results revealed that compared to control subjects, a significant lower copy number of mtDNA in the leukocytes of MetS subjects and mtDNA copy number was associated with a significant increase in HDL and inversely correlated with weight, SBP, DBP, Urate and RIFACTOR (cholesterol/HDL). MetS group had higher insulin, creatinine, urate, oxLDL, ApoB, RIFACTOR, and homocystein and lower HDL, ApoA and adiponectin in the plasma. Conclusion: The present study shows that a low mtDNA copy number of leukocytes play an important role in the progression of MetS. The results may provide new diagnostic and therapeutic strategies to MetS and other associated disorder.

碩士<br>長庚大學<br>醫學生物技術研究所<br>96<br>Mitochondrial biogenesis is regulated by the coordinated expression of nuclear genome and mitochondrial genome. Among them, three important transcriptional factors are mitochondrial transcription factor A (TFAM), nuclear respiratory factors (NRFs) and PPARγ coactivator-1α (PGC-1α). TFAM, which is localized in the mitochondria, regulates factors involving the replication and transcription of mtDNA. NRFs and PGC-1α, which are transcriptional factors in the nucleus, can interact and activate TFAM, and subsequently regulate the mitochondrial biogenesis genes. Previous studies shows that mitochondrial dysfunction could lead to cross-talk between nuclear genome and mitochondrial genome and increase the expression of mitochondrial biogenesis, but the exact mechanism of cross-talk has not been studied clearly yet. Based on this, we have used the mtDNA-depleted 143B (143B-ρ0) cell to investigate whether the defect of oxidative phosphorylation (OXPHOS) may result in alterations of expression of mitochondrial biogenesis genes, the activity of citrate synthase in tricarboxylic acid cycle (TCA cycle), and the activity of respiratory complex II. In addition, in order to exclude the possibility that the mtDNA-depletion process which might cause unknown nuclear DNA alterations could result in the changes of expression of mitochondrial biogenesis genes, we utilized the 143B-cybrid (cybrid) which was generated from 143B-ρ0 cell replenished with wild-type mtDNA for comparison to compare the alterations of mitochondrial biogenesis genes in 143B, 143B-ρ0, and cybrid cells. We used Western blot to analyze the protein levels of the genes related to mitochondrial biogenesis, employed real time RT-PCR to analyze mRNA levels of PGC-1α, and finally determined the activities of citrate synthase and complex II. The result showed that the expression of cytochrome c and ATP synthase β subunit of complex V in 143B-ρ0 cell were higher than that in 143B and cybrid cells, while levels of succinate dehydrogenase subunit B of complex II and TFAM were decreased in 143B-ρ0 cells. However, there was no difference in the level of core 1 protein of complex III among three cell lines. Taken together, we speculate that NRFs may not be the major or only factor that control mitochondrial biogenesis genes from results of Western blot analysis. Moreover, the mRNA level of PGC-1α was higher, the activity of complex II was lower, and the activity of citrate synthase was higher in 143B-ρ0 cells compared to 143B or cybrid cells. Therefore, mitochondrial biogenesis-related genes, proteins, or enzyme activities were indeed altered in mtDNA-depleted cells.

碩士<br>國立陽明大學<br>生物化學研究所<br>89<br>Abstract The pathology of mitochondrial encephalomyopathies is associated with abnormal cell loss of neurons and muscle fibers. Is it possible that mitochondrial respiratory chain dysfunction results in cell loss, which is commonly found in patients with encephalomyopathies? The respiratory chain impairment may be caused by DNA mutations in mitochondrial or nuclear genome. Among the mitochondrial DNA (mtDNA) mutations associated with mitochondrial diseases, 4977-bp deletion is the most common one. In this study, the effects of mtDNA depletion on the apoptotic behaviors of human cells were first investigated. In order to exclude the possible contributions of nuclear background on the possible effect of mtDNA itself, r0 cells derived from 143B osteosarcoma cells were used. r0 cells that are devoid of mtDNA were obtained by long-term treatment of osteosarcoma cells with ethidium bromide (EtBr). Under the treatment of staurosporine (STS), the viability, DNA fragmentation, caspase 3 activities and ROS production of r0 and parental 143B cells were compared. The results showed that r0 cells exhibited lower viability, higher caspase 3 activity and increased cytochrome c release to the cytosol. These findings suggest that mtDNA depletion exerts damaging effects on an upstream effector of caspase activation. In addition, it was also found that r0 cells were not as sensitive to the inhibitors of scavenger enzymes as 143B parental cells. Moreover, r0 cells utilize pyruvate in culture media to provide energy needed and thus produce less ROS. Taken together, these findings suggest that it is not ROS increase per se that leads r0 cells to be more susceptible to apoptosis. By using cytoplasmic fusion techniques, 4977-bp mtDNA deletion from skin fibroblasts of a CPEO patient was introduced into r0 cells and created the so-called cybrids. The cybrids containing 0% and 85% of mtDNA with 4977-bp deletion as an experimental system were used as an experimental system to investigate the effects of high proportion of 4977-bp mtDNA deletion on apoptotic behaviors of human cells. Under the treatment of STS, both the wild-type cell line 1-3-16 and the high mutant cell line 51-10 exhibited high caspase 3 activities. However, they can be distinguished by cell viability. Moreover, by the treatment of antioxidant enzyme inhibitors, such as mercaptosuccinate and 3-amino-1, 2, 4-trizole, 51-10 cells were more susceptible to apoptosis than 1-3-16 cells. Taken together, these findings suggest that high proportion of 4977-bp mtDNA deletion may reduce the ability of cells to deal with ROS and result in apoptosis or necrosis. Based on the results of this study, it is proposed that 4977-bp mtDNA deletion renders human cells more vulnerable to oxidative stress and that antioxidant therapy can be useful for patients with CPEO syndrome. On the other hand, the apoptotic effects of mtDNA depletion in vivo are different from those observed in cultured cells because of the supplementation of pyruvate and uridine in the medium. It is warranted to better understand the pathological changes of muscles and neurons in relation to the role of mtDNA depletion in patients with MERRF syndrome.

碩士<br>國立陽明大學<br>生化暨分子生物研究所<br>95<br>Mitochondria are the major energy suppliers in mammalian cells. They are also important initiators and regulators in apoptosis. Many studies have suggested mitochondrial DNA (mtDNA) alterations may affect susceptibility of human cells to apoptosis. However, whether mtDNA depletion affects the sensitivity of human cells to drug-induced apoptosis is still controversial. In this study, I used human osteosarcoma 143B cell line as the experimental model to investigate the effect of mtDNA depletion on the response of cells to doxorubicin or staurosporine-induced apoptosis and the underlying mechanisms. After treatment of cells with 0.4 mM doxorubicin for 24, 36 and 48 hours, the viability of 143B cells devoid of mtDNA (rho-zero cells) were lower than its parental cells (45.5 %, 17.9 %, 8.9 % vs. 63.5 %, 38.5 %, 28.0 %, respectively). Rho-zero cells were also more sensitive than 143B cells after treatment with 25 nM staurosporine for 8, 16 and 24 hours (78.3 %, 44.2 %, 17.9 % vs. 99.9 %, 81.6 %, 79.1 %, respectively). The proportions of rho-zero cells in sub-G1 phase (28.1 % and 45.5 %) were higher than those of 143B cells (2.8 % and 6.3 %) after treatment with doxorubicin or staurosporine. Apoptosis-related activation of caspases 8, -9, and -3 were more pronounced in rho-zero cells than that in 143B cells. As a result, rho-zero cells were more sensitive to drug-induced apoptosis as compared with 143B cells. Although the endogenous H2O2 level in rho-zero cells was lower than that in 143B cells, drug-induced H2O2 level was increased about 2 folds in rho-zero cells, which was higher than that in 143B cells. Besides, treatment of cells with exogenous H2O2 increased drug-induced apoptosis in both 143B and rho-zero cells. Therefore, reactive oxygen species (ROS) may be involved in the apoptosis of 143B cells. On the other hand, the phosphorylation level of Akt was found to be different between 143B and rho-zero cells. Upon treatment with doxorubicin or staurosporine, the phosphorylation level of Akt in 143B cells was upregulated at 4 hr and sustained to 24 hr, whereas it was remained unchanged in rho-zero cells. Inhibition of Akt phosphorylation by LY294002 could enhance doxorubicin and staurosporine-induced caspase 3 activation by 3-4 folds in 143B cells. By contrast, no such effect of LY294002 was observed in rho-zero cells. The activation of Akt may provide a protective mechanism in 143B cells, which rendered 143B cells more resistant to doxorubicin- or staurosporine-induced apoptosis. Furthermore, I reduced mtDNA content by RNA interference of the mitochondrial transcription factor A (mtTFA). The mtDNA content of 143B cells was reduced to 40 % after transfection with mtTFA siRNA for 4 days. After treatment of the cells with doxorubicin or staurosporine, no significant differences in the viability were observed between mtTFA knockdown cells and the controls. No marked differences in drug-induced H2O2 level and Akt phosphorylation were observed between mtTFA knockdown cells and the controls. This indicates that the degree of depletion of mtDNA may determine the sensitivity of 143B cells to doxorubicin and staurosporine. Taken these findings together, I suggest that ROS and Akt activation may play a role in the differential response to doxorubicin and staurosporine-induced apoptosis between 143B and rho-zero cells.

碩士<br>臺北醫學大學<br>生物醫學技術研究所<br>92<br>Down’s syndrome is the most common disease of chromosomal aberration on cytogenetics. The major form of chromosomal aberration is trisomy 21, an extra chromosome of chromosome 21. There are many studies in Down’s syndrome including clinical pathology, cytogenetics and molecular genetics. It is distinct why the germ cells are susceptible to trisomy formation at meiosis or mitosis stage. Recently, some researches reported that oxidative stress might play some roles in the formation of trisomy. Mitochondria are the major organelles that produce reactive oxygen species. In this study, we hypothesized the mitochondrial dysfunction might be contributed to the pathogenesis of Down’s syndrome. We investigated on mitochondria DNA (mtDNA) copy number, mtDNA deletion and oxidative damages in amniotic cells of Down’s syndrome patient. Decreased copy numbers of mtDNA were found in the patients of Down’s syndrome. Moreover, a novel 5335 bp mtDNA deletion was identified in amniotic cells from Down’s syndrome patient. Analysis of nucleotide sequences flanking the breakpoints of this deletion revealed a 10 nucleotides direct repeat “CCTATAGCAC” flanking the junction site of the 5335 bp deletion at nucleotide position 8263-8272 and 13598-13607. Increased ROS generation and oxidative damages were revealed in Down’s amniotic cells. Taken these data together, we suggested that the dysfunctional mitochondria might play a role in the pathogenesis of Down’s syndrome.

Mitochondrial disorders are considered to be the most common cause of metabolic abnormalities in the paediatric neurology population (Zeviani et al., 1996). These authors reported that the phenotypes observed in 25-30% of the paediatric patients in their neurology clinics were due to a mitochondrial aetiology. The genetic aetiology in an equivalently affected paediatric population in South Africa is currently unknown. This study investigated the possibility that reported mutations could account for the mitochondrial phenotypes observed in the South African population. It focussed on the most frequent paediatric mitochondrial disorders namely: Leigh Syndrome (LS), mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), and myoclonic epilepsy and ragged red muscle fibres (MERRF). A clinically well characterised group of 25 patients with mitochondrial disorders was included in this study. The molecular analysis of the mitochondrial genome was initially based on a restriction fragment length polymorphism (RFLP) screening strategy for the ten most common mitochondrial DNA (mtDNA) mutations associated with the above¬mentioned three disorders. However, during the study the mutation analysis strategy was modified to a sequencing strategy as this provided more information than the RFLP approach. The modified sequencing strategy extended the study to incorporate fifteen additional mtDNA mutations, associated with other mitochondrial disorders, and individuals included in the study were thus investigated for the presence of 25 mtDNA mutations. Moreover, the modified strategy provided additional information of the regions encompassing the reported mutations. A single patient was observed to harbour the reported A3243G MELAS mutation. This mutation was noted to be heteroplasmic in the proband and two of her maternal relatives. None of the other 24 reported mutations were observed in this patient population. One novel mtDNA alteration in the tRNALeu(UUR) gene was observed in a single patient, although the pathogenicity of this mutation remains to be investigated. Novel and reported polymorph isms, some of which are associated with specific haplogroups, were also observed when comparing sequencing data against the Cambridge reference sequence. The data generated during this study contributed towards the understanding of the uniqueness of the South African population in the global context. This was apparent from the fact that only one of the reported mutations was observed in our patient population who were clinically well characterised and displayed phenotypes similar to those reported internationally. Results form this study underlined the complexity of mitochondrial disorders and argues in favour of whole mitochondrial genome sequence information to be used for diagnostic purposes. Moreover, the results confer with the hypothesis that novel mitochondrial mutations may account for the majority of mitochondrial phenotypes observed in the South African population.<br>Dissertation (MSc (Human Genetics))--University of Pretoria, 2007.<br>Genetics<br>unrestricted

博士<br>國立陽明大學<br>生物化學研究所<br>87<br>The cellular respiratory functions decline in aging human tissues and affected tissues of patients with mitochondrial diseases. This is accompanied by an increase of reactive oxygen species (ROS) production in mitochondria via the increased electron leak of the respiratory chain. As a result, an ever-increasing amount of ROS may escape from the defense systems and cause oxidative damage to biomolecules. To investigate the relationship between free radical scavenging enzymes and oxidative damage, a total of 76 normal subjects of different ages were recruited in this study. I found an age-dependent increase of 8-OHdG content and proportion of 4,977 bp mitochondrial DNA (mtDNA) deletion in skin tissues from subjects above the age of sixty years. Moreover, I also found an age-dependent increase of lipid peroxides in cultured fibroblasts of the subjects above the age of sixty years. To evaluate the antioxidant defense system from individuals of different ages, I measured the activities of Cu,Zn-superoxide dismutase (Cu,Zn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase (CAT) and glutathione peroxidase (GPx). The activities of Cu,Zn-SOD, CAT and GPx were found to decrease with age, and the activity of Mn-SOD was increased with age before sixty years but was decreased thereafter. This indicated that elevated oxidative stress caused by an imbalance between the production and removal of ROS occurred in skin fibroblasts after sixty years of age. Taken together, I suggest that the functional decline of free radical scavenging enzymes and the elevation of oxidative stress may play an important role in eliciting oxidative damage and mutation of mtDNA during the human aging process. On the other hand, I investigated the alterations of age-dependent mitochondrial gene expression associated with mtDNA deletions. Total RNAs were isolated and analyzed by Northern hybridization. There were no significant differences of the mtRNAs among the skin fibroblasts from subjects of different ages. Moreover, I measured the activities of respiratory enzymes NCCR, SCCR and CCO in fibroblasts of subjects of different ages. There was an age-dependent decrease in the activities of the three enzyme complexes. More interestingly, I found a 75-year-old woman who had no mitochondrial disease but harbored 60% of the 4,977 bp deleted mtDNA in the fibroblasts established from her face skin. This deletion caused dramatic alteration in the mRNA levels and the activities of respiratory enzymes of mitochondria. In the second part of this study, skin and muscle fibroblasts were cultured from nine patients with chronic progressive external ophthalmoplegia (CPEO) syndrome for studying the relationship between mtDNA mutation and free radical scavenging enzymes. The skin and muscle fibroblasts from these CPEO patients carried different mtDNA mutations at different levels. The fibroblasts from patients had the common 4,977 bp mtDNA deletion, a 4,366 bp mtDNA, a 6,190 bp mtDNA deletion, a ~10 kb mtDNA deletion and the A3243G mutation in mitochondrial tRNALeu(UUR) gene. To examine whether these mtDNA mutations are associated with a defect or imbalance in the free radical scavenging enzymes. I assayed the enzyme activities of Mn-SOD, catalase and GPx of the fibroblasts. I found that the skin and muscle fibroblasts from the nine CPEO patients all had higher enzyme activity and mRNA level of Mn-SOD but those of catalase and GPx were not increased or even decreased. These results indicated an imbalance between the H2O2 generation and removal systems in the fibroblasts of the patients with CPEO syndrome. The ratios of activities of Mn-SOD/catalase in muscle fibroblasts (between 3.39 and 8.36) were about 1.4 to 3.5 fold higher than those of the control subjects. The ratios in skin fibroblasts of patients CPEO4, CPEO6, and CPEO8 were also significantly different from those of the control subjects. But the imbalance was much more pronounced in muscle fibroblasts. This tissue-specific imbalance of free radical scavengers was confirmed by analysis of the corresponding mRNA levels. The average mRNA ratio of Mn-SOD/catalase in fibroblasts of the nine CPEO patients was about 1.21 and that was 0.44 for control skin fibroblasts. By contrast, the average Mn-SOD/catalase mRNA ratio was 1.58-6.29 in muscle fibroblasts of these patients, which was much higher than those of the control fibroblasts (0.79). These results are consistent with the clinical observation that muscle is the main affected tissue of patients with CPEO syndrome. I suggest that the imbalance between free radical scavenging enzymes play an important role in the pathogenesis and age-dependent progression of the CPEO syndrome.

Der plötzliche Kindstod (engl. Sudden Infant Death Syndrome-SIDS) ist die häufigste Todesursache bei Säuglingen innerhalb des ersten Lebensjahres. Die zugrundeliegenden pathophysiologischen Veränderungen sowie die genaue Todesursache sind bis dato ungeklärt. Viele Forschungsbereiche setzen sich intensiv mit der Klärung dieses „Phänomens“ auseinander. Ein Schwerpunkt liegt auf dem genetischen Gebiet und der Betrachtung verschiedener Polymorphismen. Ein Fokus wird hierbei auf die genetischen Polymorphismen der mitochondrialen DNA (mtDNA) gesetzt. In der vorliegenden Arbeit wurden daher drei Polymorphismen der mtDNA und mögliche Risikofaktoren im Bezug zu SIDS-Fällen untersucht. Die Folge eines mitochondrialen Polymorphismus kann beispielsweise die verminderte Genexpression der Untereinheiten der Atmungskette zur Folge haben. Daraus kann ein Defizit in der ATP (Adenosintriphosphat)-Produktion resultieren. Der physiologische Kreislauf einer menschlichen Zelle ist durch dieses Defizit nur eingeschränkt gewährleistet. Im Rahmen der Forschungsarbeit wurden die SNPs G3010A, T16519C und C7028T der mtDNA in Hinblick auf einen möglichen Zusammenhang mit dem SIDS untersucht. Schon 2003 untersuchten Divne et al. (2003) einen möglichen Zusammenhang der SNPs G3010A und C7028T im Zusammenhang mit SIDS, jedoch ohne signifikantes Ergebnis. Boles et al. (2010) konnten eine Assoziation zwischen den Polymorphismen G3010A und T16519C mit dem plötzlichen Kindstod herstellen. Da bislang jedoch keine ausführliche Publikation zu dieser Frage vorliegt, wurde mit der vorliegenden Arbeit die Rolle der Polymorphismen G3010A und T16519C in Bezug auf den plötzlichen Kindstod gemeinsam mit der (bei Europäern) häufigsten Variation C7028T untersucht. Die DNA von 176 SIDS-Fällen und einer Kontrollgruppe von 113 Erwachsenen wurde mittels Singleplex-PCR und RFLP-Analyse genotypisiert. Anhand der Genotypisierung konnten die SNPs quantifiziert und im Hinblick auf einen möglichen Unterschied zwischen SIDS-Fällen und der Kontrollgruppe untersucht werden. Bei Betrachtung der einzelnen SNPs G3010A, T16519C und C7028T lassen sich keine signifikanten Unterschiede zwischen den SIDS-Fällen und der Kontrollgruppe feststellen. Das gehäufte Vorliegen einer erhöhten Mutationsrate in einem Individuum bei SIDS-Fällen im Vergleich zur Kontrollgruppe, sowie die von Opdal et al. (1999) geäußerte Annahme, dass beim Vorliegen einer Mutation in der D-Loop-Region weitere Mutationen im kodierenden Bereich vorkommen, konnten durch diese Arbeit bestätigt werden.

Tese de doutoramento em Ciências da Saúde, no ramo de Ciências Biomédicas, apresentada à Faculdade de Medicina da Universidade de Coimbra<br>Myeloid neoplasms (MN) are a group of heterogeneous diseases that includes myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and acute myeloid leukemia (AML). These diseases can simultaneously harbor changes in reactive oxygen species (ROS) levels and in DNA methylation pattern. The oxidative stress (OS) results from the imbalance between ROS production, mainly in mitochondria, and their elimination by antioxidants. Moreover, MN recurrently show abnormal hypermethylation of tumor suppressor genes (TSG), such as P15 and P16 genes, and in less extent global hypomethylation of repetitive sequences such as long interspersed nucleotide elements 1 (LINE-1). The present study aimed to analyze the involvement of OS and DNA methylation in the development and progression of MN, in order to evaluate their role as diagnostic and prognostic markers, as well as to identify susceptibility variants in genes involved in these mechanisms. Frist, we conducted a proof of concept study to investigated the involvement of OS and mitochondrial dysfunction in MDS pathogenesis, as well as to assessed their diagnostic and prognostic value, and the relation of OS parameters (intracellular levels of peroxides, superoxide, and reduced glutathione – GSH) with methylation status of P15 and P16 gene promoters. To that end we used two technique: flow cytometry and methylation specific-PCR (MSP). We observed that bone marrow cells from MDS patients had higher peroxide levels and lower GSH content than control cells. Moreover, OS levels were dependent of MDS subtype and risk group. GSH showed to be an accurate MDS diagnostic marker, while ROS, GSH, and superoxide/peroxides ratio were good survival markers. MDS patients had higher P15 and P16 methylation frequencies than controls. Moreover, patients with methylated P15, P16, and P15 or P16 had higher OS levels than patients without methylation. To confirm these results, we expanded the evaluation of OS (several enzymatic and non-enzymatic antioxidant defenses; oxidative damage) and DNA methylation parameters [localized DNA methylation – methylation status of P15, P16, TP53, MGMT, DAPK, and KEAP1 genes; global DNA methylation – levels of 5-methylcytosine (5-mC), 5-hydroxymetylcytosine (5-hmC), and LINE-1 methylation], as well as the studied pathologies (MDS and MPN). These biological parameters were evaluated in peripheral blood samples by colorimetry and fluorimetry assays, as well as by MSP and combined bisulfite restriction analysis (COBRA). MDS patients had lower levels of GSH and TAS (total antioxidant status), as well as higher levels of peroxide, peroxide/GSH, and peroxide/TAS than controls. Moreover, MDS and MPN patients had higher 5-mC levels and lower 5-hmC/5-mC ratio, as well as increased methylation of at least one methylated gene (P15, P16, DAPK, or KEAP1). TP53 and MGMT genes were unmethylated in these patients. Peroxide levels and peroxide/GSH ratio were higher in patients with methylated genes than in those without methylation. LINE-1 methylation and 5-mC levels were correlated with peroxide levels and peroxide/GSH ratio. Next, we assessed the association of variants of genes involved in OS, folate metabolism, DNA repair, and DNA methylation with the susceptibility and prognosis of MDS and AML. To that end, 16 SNPs (one per gene: CAT, CYBA, DNMT1, DNMT3A, DNMT3B, GPX1, KEAP1, MPO, MTRR, NEIL1, NFE2F2, OGG1, SLC19A1, SOD1, SOD2, and XRCC1) were genotyped by PCR techniques. We also analyzed OS (ROS/TAS), DNA damage (8-OHdG), and DNA methylation (5 mC) in a sub-cohort of MDS patients and controls. Results showed that five genes (GPX1, NEIL1, NFE2L2, OGG1, and SOD2) were associated with MDS, two (DNMT3B and SLC19A1) with AML, and two (CYBA and DNMT1) with both diseases. OS levels were correlated with CYBA, GPX1, and SOD2 genotypes, DNA damage with NEIL1 and OGG1, and 5-mC levels with DNMT1, DNMT3A, DNMT3B, and MTRR. Furthermore, DNMT3A, MTRR, NEIL1, and OGG1 variants modulated AML transformation in MDS patients. Additionally, DNMT3A, OGG1, GPX1, and KEAP1 variants influenced MDS and AML survival. Finally, we investigated if acute and chronic exposure to hydrogen peroxide (H2O2) affects the methylome of normal and malignant hematological cells. In this investigation, we used four acute myeloid leukemia cell lines and a normal B lymphocyte cell line, and analyzed the copy number and methylation status of several TSG, LINE 1 methylation, levels of 5-mC, 5-hmC, 8-hydroxy-2'-deoxyguanosine (8-OHdG), ROS, and GSH, as well as the gene expression of DNMT1, DNMT3A, DNMT3B, MECP2, MBD1, HDAC1, EZH2, EP300, and TET2. These analyses were performed by methylation-specific multiplex ligation dependent probe amplification (MS-MLPA), COBRA, colorimetry and fluorimetry assays, as well as real time PCR. Acute and chronic exposure to H2O2 increased TSG methylation (in cells with increased ROS/GSH ratio) and decreased LINE-1 methylation (in cells with increased GSH levels). TSG hypermethylation was cell line-dependent and accompanied by upregulation of DNMT1, DNMT3A, MECP2, HDAC1, and EZH2 genes. Moreover, the pre-treatment with N acetylcysteine, an antioxidant molecule, prevented these events. Overall, the present study points to a possible link between OS and DNA methylation, which besides the relevance in the development and progression of these MN, could also constitute new diagnostic and prognostic markers as well as new potential therapeutic targets.<br>As neoplasias mieloides (NM), de que são exemplo a síndrome mielodisplásica (SMD), as neoplasias mieloproliferativas (NMP) e a leucemia mieloide aguda, são um grupo heterogéneo de doenças clonais hematopoiéticas. A patogénese destas doenças pode envolver simultaneamente alterações nos níveis de stresse oxidativo (SO) e no padrão de metilação do ADN. O SO resulta do desequilíbrio entre a produção de espécies reativas de oxigénio (ROS), geradas principalmente na mitocôndria, e a sua eliminação pelas defesas antioxidantes. Além disso, estas doenças apresentam frequentemente hipermetilação nos genes supressores de tumor (GST), como os genes P15 e P16, e alguns casos podem também apresentar hipometilação nas sequências repetitivas, nomeadamente as long interspersed nucleotide elements 1 (LINE-1). O presente estudo teve como objetivo analisar o envolvimento do stress oxidativo e da metilação do DNA no desenvolvimento e na progressão das neoplasias mieloides, de modo a avaliar o seu papel como marcadores de diagnóstico e prognóstico, e identificar variantes genéticas de susceptibilidade em genes envolvidos nesses mecanismos. Inicialmente, efetuou-se um estudo de prova de conceito de modo a avaliar o envolvimento do SO e a disfunção mitocondrial na patogénese das SMD, o seu valor como marcadores de diagnóstico e de prognóstico, e a relação dos parâmetros OS (níveis intracelulares de peróxidos, anião superóxido e glutationa reduzida – GSH) com o perfil de metilação dos promotores dos genes P15 e P16. Para este efeito, recorreu-se a duas técnicas: citometria de fluxo e PCR específico de metilação (MSP). Os resultados demonstram que as células de medula óssea dos doentes SMD apresentam aumento dos níveis de peróxidos e diminuição de GSH, relativamente às células dos controlos. No entanto, estes resultados dependem do subtipo de SMD e do grupo de risco. Além disso, a GSH revelou-se um bom marcador de diagnóstico, enquanto os ROS, a GSH e a razão superóxido/peróxidos mostraram-se marcadores de sobrevivência. Estes doentes apresentam frequências de metilação dos genes P15 e P16 mais elevadas do que os controlos. Por outro lado, os doentes SMD com metilação nos gene P15, P16, e P15 ou P16 apresentam níveis de SO mais elevados do que os doentes sem metilação. De modo a confirmar estes resultados, realizou-se uma avaliação alargada dos parâmetros de SO (várias defesas antioxidantes enzimáticas e não enzimáticas; lesão oxidativa) de metilação do DNA [metilação localizada – perfil de metilação dos genes P15, P16, TP53, MGMT, DAPK e KEAP1; metilação global – níveis de metilação da 5-metilcitosina (5-mC), da 5-hidroximetilcitosina (5-hmC) e da LINE 1] não só em doentes SMD, mas também em doentes NMP. Estes parâmetros biológicos foram avaliados em amostras de sangue periférico por colorimetria, fluorimetria, MSP e COBRA (combined bisulfite restriction analysis). Os resultados demonstraram que os doentes SMD apresentam diminuição dos níveis de GSH e CAP (capacidade antioxidante total), assim como aumento dos níveis de peróxido e das razões peróxido/GSH e peróxido/CAP comparativamente aos controlos. Por outro lado, observou-se aumento dos níveis de 5-mC e diminuição da razão 5-mC/5-hmC nos doentes SMD e NMP. Além disso, este doentes apresentam aumento da frequência de metilação de pelo menos num gene (P15, P16, DAPK, ou KEAP1). No entanto, não se observou metilação dos genes TP53 e MGMT nestes doentes. Os doentes genes metilados apresentam aumento dos níveis de peróxido e da razão peróxido/GSH relativamente aos doentes sem metilação. Por fim, os níveis de metilação da LINE-1 e da 5-mC correlacionam-se com os níveis de peróxido e da razão peróxido/GSH. Seguidamente, avaliou-se os polimorfismos em genes envolvidos no SO, no metabolismo do folato, na reparação e na metilação do DNA e o seu papel na susceptibilidade e prognóstico dos doentes SMD e LMA. Com essa finalidade, genotiparam-se 16 SNPs (um por gene: CAT, CYBA, DNMT1, DNMT3A, DNMT3B, GPX1, KEAP1, MPO, MTRR, NEIL1, NFE2F2, OGG1, SLC19A1, SOD1, SOD2 e XRCC1) por técnicas de PCR. Analisaram-se, igualmente, os níveis de OS (ROS/CAP), da lesão (8-OHdG) e da metilação do DNA (5-mC) num subgrupo de doentes SMD e controlos. Os resultados sugerem que os genes GPX1, NEIL1, NFE2L2, OGG1 e SOD2 influenciam a susceptibilidade para SMD, os genes DNMT3B e SLC19A1 a susceptibilidade para LMA e os genes CYBA e DNMT1 o desenvolvimento das duas doenças. Além disso, os níveis de SO correlacionam-se com o genótipo dos genes CYBA, GPX1 e SOD2, a lesão do DNA com os genes NEIL1 e OGG1, e os níveis de 5-mC com os genes DNMT1, DNMT3A, DNMT3B e MTRR. Os polimorfismos nos genes DNMT3A, MTRR, NEIL1 e OGG1 influenciam o prognóstico (evolução para LMA) dos doentes SMD, enquanto os polimorfismos nos genes DNMT3A, OGG1, GPX1 e KEAP1 influenciam a sobrevivência dos doentes SMD e AML. Por fim, investigou-se a influência da exposição crónica e aguda ao peróxido de hidrogénio (H2O2) na metilação de células hematológicas normais e malignas. Para o efeito, utilizaram-se quatro linhas celulares de leucemia mieloide aguda e uma linha celular de linfócitos B normal. A variação do número de cópias e o perfil de metilação de vários GST, os níveis de metilação da LINE-1, da 5-mC, 5-hmC, da 8-hidroxi-2'-desoxiguanosina (8-OHdG), das ROS e da GSH, bem como a expressão dos genes DNMT1, DNMT3A, DNMT3B, MECP2, MBD1, HDAC1, EZH2, EP300 e TET2 foram avaliados por methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), COBRA, colorimetria, fluorimetria e PCR em tempo real. A exposição aguda e crónica ao H2O2 induziu hipermetilação nos GST (nas células com aumento da razão ROS/GSH) e hipometilação nas sequências LINE-1 (nas células com aumento da GSH). A hipermetilação dos GST foi acompanhada da sobre-expressão dos genes DNMT1, DNMT3A, MECP2, HDAC1 e EZH2 de modo dependente da linha celular. Além disso, o pré-tratamento com N-acetilcisteína, uma molécula antioxidante, preveniu estas alterações moleculares. Em conclusão, o presente estudo sugere uma possível relação entre o stresse oxidativo e a metilação do DNA, os quais, além da relevância no desenvolvimento e na progressão das neoplasias mieloides, poderão igualmente constituir novos marcadores de diagnóstico e prognóstico, bem como potenciais alvos terapêuticos.<br>Fundação para a Ciência e Tecnologia