Thèses sur le sujet « Mitochondrial disease, gene discovery »

My researches during the DIMET project have been focused on the discovery of new genes responsible for mitochondrial disorders and the characterization of their role. Recent epidemiological studies show that mitochondrial disorders have an incidence of 1:5000. These disorders are very heterogeneous and hence the diagnosis is difficult. Moreover mitochondrial dysfunctions are now clearly related to a wide range of disease conditions (i.e. neurodegeneration and cancer). The majority of the inherited mitochondrial disorders, especially those with onset in infancy or childhood, are due to nuclear genes encoding proteins targeted to mitochondria. While identification of mutations in mitochondrial DNA has become relatively easy thank to the feasibility to perform the complete sequence analysis of mtDNA, the analysis of genomic DNA is more complicate and therefore the number of nuclear genes associated with mitochondrial diseases is still small. Genome-wide analysis in families with autosomal recessive mitochondrial disorders could help to identify a genomic region to be further investigated. However, about one half/one third of the components of the mitochondrial proteome have yet to be identified, and this lack of information makes the search of candidate genes more difficult. By linkage analysis or homozygosity mapping and prioritization of candidate genes, I studied subjects from multiconsanguineos families characterized by clinical pictures compatible with mitochondrial disorders. In chapter 2, there is the report regarding the discovery of a nonsense mutation in two brothers displaying asymmetric brain atrophy, psychomotor regression and severe complex IV deficiency. The mutated gene codes for a mitochondrial predicted kinase that may have a role in apoptosis. Using the same procedure, I take part in a project, which leads to the identification of the first assembly factor for complex II of the OXPHOS system (Chapter 3). Two different mutations were found in two pedigrees, with affected children characterized by acute psychomotor regression followed by spastic quadriparesis and/or dystonia. The pathogenic role of the mutations was confirmed in cellular and yeast models. Finally, in chapter 4, there is the characterization of a protein, MR-1, already known and responsible for a movement disorder (PNKD, Paroxysmal non kinesigenic Dyskinesia). The mutant isoforms were erroneously localized into cytosol or membranes, whereas I demonstrated that they are mitochondrial and that the mutations reported so far in PNKD patients (and a new mutation identify in our study) are in the mitochondrial targeting signal (MTS). Hence PNKD could be considered a mitochondrial disease, due to a novel mechanism based on a deleterious action of the MTS.

Although mitochondrial disorders are the most common inherited form of neuromuscular disease, there are currently limited effective treatments that directly improve mitochondrial function – either by modulation of the effects of mutated genes or by increasing the proportion of healthy mitochondria. In this work, two different approaches were employed to develop treatments for mitochondrial diseases: the design of mitochondrially targeted anti-sense oligonucleotides and the development of a high throughput screen of a unique library of bacterial extracts. The heteroplasmic nature of mitochondrial DNA (mtDNA) enables the use of anti-genomic strategies to specifically prevent the replication, transcription or translation of mutated molecules of mtDNA or mitochondrial mRNA in patients with heteroplasmic mtDNA mutations. In conjunction with an industrial partner (Ugichem GmbH), a mitochondrial targeting oligonucleotide was developed using cell membrane crossing oligomers (CMCOs) – a new class of oligonucleotide with the ability to enter and accumulate within the cytoplasm. By conjugating “mitochondrial targeting” molecules to the CMCOs, translocation to mitochondria was shown, potentially enabling the use of anti-sense therapies in the treatment of mtDNA diseases. To complement the mutation specific approach of anti-sense oligonucleotide therapies a large scale screen was carried out to discover compounds that could cause a general improvement in mitochondrial function. A library of unique bacterial extracts, provided by Demuris Ltd, was screened for effects on mitochondrial biogenesis in HeLa cells. To that end, a high throughput assay, which used fluorescent markers to detect changes in relative mitochondrial mass, was designed and validated using mitochondrially active control compounds. The screen of bacterial extracts discovered several one extract which caused mitochondrial mass to increase 2 fold after 48 hours incubation.

Mitochondrial respiratory chain disorders (MRCD) are one of the most challenging inborn errors of metabolism to diagnose and treat, because of their phenotypic and genetic heterogeneity, and a paucity of validated therapies. The approach opted to diagnose our MRCD patient cohort was using next generation sequencing (NGS) technology, and in particular whole exome sequencing (WES). Using our research pipeline we were able to identify novel YARS2 variants in patients with a tissue specific MRCD. Novel disease gene discoveries (CYC1 and MRPS7) causing MRCD were identified using the WES approach, and the functional studies implemented confirmed the pathogenicity of the newly identified variants. Advances in NGS technologies lead to the identification of a novel mtDNA mutation in the MT_ND3 gene and therefore rapid genetic diagnosis of a patient with Leigh disease allowing timely acceptance into a Phase 2B clinical trial. Using WES, two patients initially suspected to have a mitochondrial myopathy were diagnosed with congenital myasthenia resulting in the successful treatment for one patient. Through the research presented in this thesis, we were able to gain more knowledge about the disease mechanisms of MRCDs, and we hope that these findings will be instrumental in research towards future therapy of MRCD.

Congenital myopathy and muscular dystrophy are two groups of inherited muscle diseases characterised by muscle weakness, and sub-classified by hallmark pathological features within a skeletal muscle biopsy. In order to understand the pathogenesis of inherited muscle disorders, and develop or apply therapies based on mechanistic insight, one must elucidate deep knowledge of the associated gene, genetic variant and the function of the encoded protein. This thesis focuses on three aspects of gene discovery in the inherited myopathies: (1) Identification of a novel variant and phenotype for a known disease gene; (2) understanding the functional role of a recently identified disease gene in skeletal muscle biology and disease; and (3) discovering a novel disease gene for congenital myopathy. We identified the first recessive variant within ACTA1 (encoding α-skeletal actin) as the genetic cause of congenital muscular dystrophy with rigid spine. This case uniquely describes recessive ACTA1 variants where α-skeletal actin protein is expressed. The unique clinical and histological presentation expands the spectrum of ACTA1 disease, and will help guide clinical care and future genetic diagnoses. Our team identified LMOD3 (leiomodin-3) as a novel disease gene for severe nemaline myopathy (NM). KLHL40 (encoding kelch-like family member 40) is another disease gene for severe NM. A recent study suggests mouse Klhl40 protects mouse Lmod3 protein from proteasome-mediated degradation, with the mechanistic basis of KLHL40-NM resulting from secondary loss of LMOD3. We investigated the regulation of human LMODs by human KLHL40, and unexpectedly found evidence that disputes the central paradigm that KLHL40 protects LMOD3 from proteasome-mediated degradation. We identified PYROXD1 as a new genetic cause of early-onset congenital myopathy. We provide the first characterisation of PYROXD1 as a nuclear-cytoplasmic oxidoreductase and our discovery highlights oxidative distress as a core mechanistic pathway in the myopathies. We derived a mouse model of Pyroxd1 deficiency, determining that global loss of mouse Pyroxd1 is embryonic lethal. We subsequently developed a mouse model with skeletal muscle knock-out of Pyroxd1 – as a means to elucidate the role of PYROXD1 in biology and disease.

Les mutations de l'ADN mitochondrial sont une cause importante de maladies neuromusculaires humaines incurables. Parmi plusieurs mutations répertoriées, plus de 170 sont localisées dans les gènes codant pour les ARN de transfert (ARNt), dont 29 - dans le gène de l’ARNtLeu(UUR) (MT-TL1). La mutation m. 3243A>G de MT-TL1 a été décrite comme une cause majore du syndrome MELAS (myopathie, encéphalopathie, acidose lactique, accidents vasculaires cérébraux). Cette mutation réduit le niveau d’aminoacylation de l’ARNtLeu(UUR) et mène à l’hypomodification de la position « wobble » de son anticodon, ce qui cause des déficiences de la synthèse protéique dans l'organite et une réduction des activités des complexes de la chaîne respiratoire. L’objectif principal de la thèse a été d’étudier si l’expression des ARNt recombinants dans le noyau de cellules transmitochondriales humaines ayant la mutation MELAS m. 3243A>G et leurs adressage dans les mitochondries puisse améliorer les fonctions mitochondriales des cellules touchées par la mutation. Il a été démontré que l'expression des ARNt recombinants est accompagnée par une amélioration de la synthèse protéique mitochondriale, une augmentation du niveau des protéines codées par l’ADNmt et une restauration partielle de la respiration. Ces résultats démontrent la possibilité d’adresser dans les mitochondries les ARNt au potentiel thérapeutique dont la spécificité d’aminoacylation a été changée, et étendent ainsi l’utilisation de l’approche de l’expression allotopique pour le développement de thérapie génique des maladies mitochondriales
Mutations in human mitochondrial DNA are often associated with incurable human neuromuscular diseases. Among these mutations, more than 170 have been identified in tRNA genes, including 29 in the tRNALeu(UUR) gene (MT-TL1). The m. 3243A>G mutation in MT-TL1 was described as the major cause of the MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes). This mutation reduces tRNALeu(UUR) aminoacylation level and leads to a hypomodification of the wobble position of its anticodon, which results in a decreased level of mitochondrial protein synthesis and reduced activities of respiratory chain complexes. The thesis was aimed to test if the allotopic expression of recombinant leucine tRNAs in the nucleus of transmitochondrial cybrid cells carrying MELAS m. 3243A>G mutation and their subsequent targeting into mitochondria can rescue mutation-induced dysfunctions. It was shown that expression of specifically designed recombinant tRNAsLeu is accompanied by a significant improvement of mitochondrial translation, an increase of steadystate level of several mtDNA-encoded protein subunits of respiratory chain, and a partial rescue of respiration. These findings prove the possibility to direct into mitochondria tRNAs with changed aminoacylation specificity possessing potential therapeutic activity, thus extending the potential of allotopic expression as the approach to cure mitochondrial disorders

Purpose: Inherited retinal dystrophy (IRD) describes a collection of degenerative retinal disorders, with a prevalence of approximately 1 in 3500. Many of the underlying disease genes and their functions are not known. There is progressive degeneration of the photoreceptors which may result in complete blindness at early ages of life. Although mutations in over 250 genes have been described in IRD, diverse clinical features and the genotypic heterogeneity make the application of conventional sequencing methods very limited. The aims of this study were to: identify the underlying disease-causing gene in a cohort of 12 retinitis pigmentosa (RP) families using genomic approaches and targeted gene analysis; identify the underlying candidate disease gene in a family with a novel syndromic retinal dystrophy and investigate the function of the gene using cell-based assays; and to characterise a mouse model of the novel retinal dystrophy syndrome. Methods: Whole exome sequencing using the TruSeq exome enrichment system (Illumina Inc., San Diego, CA, USA), was applied to 12 probands diagnosed with autosomal dominant retinitis pigmentosa (ADRP, n=6) and autosomal recessive retinitis pigmentosa (ARRP, n=6). Pathogenicity was predicted and detected variants were confirmed and segregation determined using Sanger sequencing. In a family with an inherited novel syndromic retinal dystrophy described in the thesis, genomic, cell-based, and animal model approaches were undertaken to identify and understand the function of the novel disease gene. Functional studies were performed in mutant and control human fibroblasts, and transfected HeLa cells. A CRISPR/Cas9 generated mouse model of the disease was investigated using electroretinography (ERG), histology, and immunohistochemistry studies to understand the impact of the detected variant. Results: Pathogenic variants were identified in 4 families with ADRP families. Clear-cut variants were identified in 2 families with ARRP. A novel candidate retinal disease gene, alpha kinase-1 (ALPK1), has been identified in the newly described ROSAH syndrome which analysis from this thesis implicates in centrosome and cilia biology. Analyses in mouse retina showed expression of Alpk1 in the connecting cilium region of the photoreceptors with a possible role in ciliary trafficking. Cell-based assays revealed ALPK1 localisation in the centrosomes and the basal body of the primary cilium. Immunocytochemistry on skin fibroblasts revealed a higher percentage of cells with abnormal numbers of centrosomes in affected individuals. In HeLa cells transfected with the mutant construct, there were a higher number of multinucleated cells, further suggesting abnormality of centrosome biology. ERG studies revealed significant decrease in scotopic and photopic responses in mice with the orthologous mouse mutation, and histology sections showed thinning of the retinal layers. Further immunohistochemistry studies confirmed significant decrease in Tcp1 (ALPK1 binding partner) expression in the inner segment of photoreceptors and aberration of connecting cilium proteins, Ift88 and centrin in the mouse model. Conclusions: Genomic and functional work in this thesis has led to new knowledge with implications for regulation of centrosome biology and impact on ciliary function in the retina. This study highlights benefits of genomic investigation, phenotype-based bioinformatics, and functional studies to gain insight to the pathophysiology of the disease, paving the way for development of therapeutic strategies in the future.

Over the past two decades, significant understanding of the pathogenesis of Parkinson's disease (PD) has been attributed to the discovery of genes, that when mutated, are responsible for familial forms of PD. Recently a novel autosomal dominant mutation (AD) causing PD was identified in receptor-mediated endocytosis-8 (RME-8). When mutated, symptoms of PD manifest with an onset ~ 70 years of age. RME-8 is a DnaJ domain containing protein that plays an important role in intercellular trafficking and recycling of retrograde cargo. Loss of function of RME-8 disrupts the endosome to Golgi transport resulting in cargo accumulation in the endosome and its re-routing to the lysosome for degradation. Studies have shown that VPS35 (another AD-PD gene, and part of the retromer that RME-8 interacts with) is involved in the mitochondrial quality control pathway implicated in PD. In addition, recent studies have shown that bec-1, a protein long studied as a regulator of autophagy (part of the mitochondrial quality control pathway), to also be involved in the retrograde trafficking co-localizing with RME-8. These findings suggest a possible new role of RME-8 in the mitochondrial quality control pathway. Here we investigated the possible role of RME-8 in the mitochondrial quality control pathway implicated in PD. Using loss of function approach by knocking-down RME-8 and gain of function approach by overexpressing the mutant form of RME-8 we investigated its role in two pathways involved in mitochondrial quality control: mitophagy and mitochondrial vesicle formation. Our results show that RME-8 is not involved in either pathways and thus the exact role of RME-8 in the pathogenesis of PD has to still be elucidated.
Des avancées significatives dans la compréhension de la pathologie propre à la maladie de Parkinson (MP) ont marqués les deux dernières décennies grâce, notamment, à la découverte de mutations génétiques responsables de formes familiales de la MP. Récemment, une mutation autosomale-dominante (AD) dans le gène RME-8 (receptor-mediated endocytosis-8) a été identifiée comme cause de la MP dont les manifestations cliniques associées à cette mutation apparaissent vers 70 ans. La protéine codée par RME-8, contient un domaine DnaJ qui joue un rôle important dans le trafic intracellulaire et le recyclage de cargos rétrogrades. La protéine RME-8 est exprimée dans plusieurs tissus et possède une forte affinité pour la chaperonne HSC70 (heat shock protein 70). RME-8 recrute HSC70 aux membranes couvertes de clathrine et interagit avec le complexe du retromère pour désassembler les triskelions de clathrine. La perte de fonction de RME-8 perturbe le transport de l'endosome au Golgi, ce qui entraîne l'accumulation du cargo dans l'endosome et sa redirection vers le lysosome. De plus, il a été démontré, que VPS35, fait partie du complexe du retromère et interagit avec RME-8, et que BEC-1 est impliquée dans le trafic rétrograde et que l'appauvrissement de RME-8 ou BEC-1 donne des phénotypes similaires. Puisque VPS35 et BEC1 jouent un rôle dans le contrôle de la qualité mitochnodriale, nous avons émis l'hypothèse que RME-8 est aussi impliquée dans ce processus. Ni l'ablation de RME-8 via l'ARN interférence ou sa surexpression n'a permis de montrer un rôle pour RME-8 dans la mitophagie ou la formation de vésicules mitochodriales. Nos données tendent à montrer que RME-8 n'est pas impliquées dans le contrôle de la qualité mitochondriale et que son rôle dans la pathogénèse de la MP demeure obscur.

Globally, approximately 35.6 million people live with dementia, with a yearly incident increase of approximately 7.7 million. Factoring in the aging population and increasing life expectancies, current projections predict that by 2050, global prevalence of dementia will reach 155 million. Alzheimer disease (AD) is the most common cause of dementia accounting for 60-80% of cases. AD is a complex and genetically heterogeneous condition. Most cases are the result of multifactorial inheritance with advancing age being the greatest contributor to risk; however, approximately 5% of AD occurs in the context of a dominant family history. Because there is such a strong association between young onset age (before age 60-65) and dominantly inherited AD, it is unclear how often late-onset Alzheimer disease (LOAD) is due to single gene inheritance. We hypothesize that LOAD in multi-incident families is, at times, caused by single gene mutation, in either the 3 genes known to cause early-onset AD (PSEN1, PSEN2 and APP) or genes not previously associated with AD. Family history data from attendees of a referral-based memory disorder clinic were entered into a new comprehensive database which allowed selection of thirteen families suggestive of dominantly inherited LOAD. A targeted gene panel containing the coding region of 177 genes implicated in dementia and other neurodegenerative conditions was used to screen for pathogenic mutations in our candidate families. We identified 97 missense variants and 1 nonsense variant, including mutations in PSEN1 (p.I437V), PSEN2 (p.S130L), DNAJC13 (p.N855S), DCTN1 (p.T147A) and LMNA (p.N459S). Our findings justify offering diagnostic genetic testing to individuals symptomatic for LOAD with family histories suggestive of autosomal dominant inheritance. Currently, such testing is only offered to individuals with early-onset disease. This research also provides a useful framework for ongoing gene discovery in LOAD and other dementias utilizing the family history database and population-based DNA samples available at the University of British Columbia Hospital Clinic for Alzheimer Disease and Related Disorders (UBCH-CARD).
Medicine, Faculty of
Medical Genetics, Department of
Graduate

Thesis (MScMedSC)--Stellenbosch University, 2012.
Bibliography
ENGLISH ABSTRACT: Parkinson’s disease (PD) is a neurodegenerative movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra of the midbrain. Although the aetiology of PD is still not fully understood, it is thought to involve a combination of environmental (such as exposure to pesticides and neurotoxins) and genetic factors. A number of PD-causing genes have been found including SNCA, LRRK2, EIF4G1 and VPS35 (for autosomal dominant forms of PD) and parkin, PINK1, DJ-1 and ATP13A2 (for autosomal recessive forms of PD – arPD). Mutations in the parkin gene are the predominant cause of arPD. Parkin plays a role in the ubiquitin-proteasomal system which degrades damaged and unwanted proteins in the cell and it is also thought to be involved in maintaining healthy mitochondria. Numerous studies have implicated mitochondrial function in the pathogenesis of PD. Therefore the aim of the present study was to investigate the role of mitochondrial dysfunction in PD patients with parkin-null mutations. Four South African PD patients, each harbouring two parkin-null mutations, were recruited for this study. A muscle biopsy was performed for analysis of mitochondrial morphology using histology and transmission electron microscopy (TEM). Skin biopsies were taken, from which fibroblasts were cultured. These fibroblasts were used in i) mitochondrial morphological assessments using TEM, ii) mitochondrial network analysis, iii) functional studies via ROS measurement and iv) analysis of the proteome using a LTQ Orbitrap Velos mass spectrometer. In addition, RNA was isolated from peripheral blood samples for gene expression studies using the RT² Profiler PCR Array (SABiosciences, USA) and the RT² PCR Primer Assay (SABiosciences, USA). Heterozygous family members (carriers) and wild-type controls were also recruited for this study. Results from the histological and TEM analysis from the muscle biopsy observed subtle mitochondrial changes including the presence of type II fibres, atrophic fibres, the presence of lipids, and wrinkling of the sarcolemmal membrane. Enlarged mitochondria were also observed in one patient. TEM analysis on the patient’s fibroblasts observed an increase in the number of electron dense vacuoles, speculated to be autolysosomes. The mitochondrial network in two of the patients’ fibroblasts showed fragmented and dot-like networks which are indicative of damaged mitochondria. An increase in mitochondrial ROS levels was observed in three of the four patients. Expression studies found down-regulation of 14 genes from four of the five mitochondrial complexes and a total of 688 proteins were found only in the control and not in the patient fibroblasts. Some of these proteins are known to be part of the ‘mitochondrial dysfunction’ pathway. Taken together, these results indicate that the absence of parkin results in a number of mitochondrial alterations. Based on these findings, a model of PD was proposed: It is speculated that when parkin is absent, electron transport chain complex genes are down-regulated. This results in impaired oxidative phosphorylation, causing an increase in the production of mitochondrial ROS and subsequent oxidative stress. Mitochondria are then damaged; resulting in the fragmentation of the mitochondrial network. The impaired mitochondria are thus tagged for degradation, causing the recruitment of autolysosomes which engulf the mitochondria via mitophagy. Ultimately, as the compensatory mechanisms fail, this triggers the consequential cascade of cellular apoptotic events. This study has elucidated the effect of parkin on the mitochondria, and can act as a ‘stepping stone’ towards future development of therapeutic strategies and/or biochemical markers that will benefit not only patients with PD but also other neurodegenerative disorders.
AFRIKAANSE OPSOMMING: Parkinson se siekte (PS) is ‘n neurodegeneratiewe bewegings-afwyking gedefineer deur die verlies van dopaminergiese neurone in die substantia nigra van die midde brein. Alhoewel die spesifieke oorsprong van die afwyking nog nie ten volle begryp is nie, word bydraes van beide omgewings faktore (bv. blootstelling aan plaagdoders en neurotoksienes) asook genetiese faktore gespekuleer. Vanuit ‘n genetiese aspek is ‘n aantal gene al geassosieer met PS. Hierdie gene sluit in SNCA, LRRK2, EIF4G1 en VPS35 (vir outosomale dominante vorms van PS) en parkin, PINK1, DJ-1, en ATP13A2 (vir outosomale resessiewe vorms van PS - orPS). Mutasies in die parkin geen is aangedui as die hoof oorsaak van orPS. Parkin speel ‘n rol in die ubiquitine-proteasomale sisteem wat beskadige en ongewensde proteïne binne in die sel verwyder en is verdink om by te dra tot die instandhouding van gesonde mitokondria. Mitokondriese wanfunksionering is ook deur talle studies gewys as ‘n bydraende faktor in die patologie van PS. Die doel van die studie is om ondersoek in te stel tot die spesifieke rol wat mitokondriese wanfunsionering speel in PS pasiënte met parkin-nul mutasies. Vier Suid-Afrikaanse PS-pasiënte, elk met twee parkin-nul mutasies, is gebruik vir die studie. Deur middel van spierbiopsies is monsters verkry vir mitokondriese morfologiese analises met behulp van histologiese en elektron-oordrag mikroskopie tegnieke (TEM). Vel biopsies is ook geneem en fibroblaste is gekweek vir die gebruik in: i) mitokondriese morfologiese assesering; ii) mitokondriese netwerk analiese; iii) funksionele studies waar vlakke van reaktiewe suurstof spesies (ROS) gemeet is; iv) proteoom analiese met behup van ‘n LTQ Orbitrap Velos massa spektrometer. RNA is ook geisoleer vanaf perifere bloedmonsters vir die gebruik in geen-uitdrukkings studies met behulp van ‘n RT² Profiler PCR Array en ‘n RT² Primer Assay. Selle vanaf famielie lede wat heterosigotiese draers is van die mutasie, asook normale (geen parkin mutasie) selle is gebruik as kontroles in die studie. TEM resultate vanaf die spier monsters het subtiele mitokondriese veranderinge getoon. Hierdie sluit in die teenwoordigheid van tipe II vesels, atrofiese vesels, teenwoordigheid van lipiedes, assook waarnemings van rimpeling van die sarcolemmal membraan. Vergrote mitokondrias is ook in een van die pasiënte opgelet. TEM resultate vanaf die fibroblaste het toename in die aantal elektron-digte vakuole vertoon, moontlik geidentifiseer as autolisosome. Gefragmenteerde en onderbreekte mitokondria netwerke is gelet tydens netwerk analiese van die fibroblaste, ‘n indikasie van beskadigde mitokondria. ‘n Toename in mitokondriese ROS vlakke is gevind in drie van die vier pasiënte. Af-regulering van 14 gene, geassosieerd met vier uit die vyf mitokondria komplekse, is verneem tydens die geen-uitdrukkings studie. Saam met dit is ‘n totaal van 688 proteïene geidentifiseer wat slegs teenwoordig is in die kontrole monsters en nie in die pasiënt monsters nie. Hierdie proteïene is almal uitgedruk en betrokke in die mitokondriese wanfunsionerings-weë. Hierdie resultate dui dat die afwesigheid van parkin mitokondriese afwykings tot gevolg het wat kan lei tot die afsterwing van selle. Dit dra ook by tot die vorming van ‘n beter-verstaande siekte-model vir PS: Mutasies in parkin (wat lei tot die afwesigheid van parkin) kan dus moontlik lei tot die af-regulasie van gene geassosieerd met die elektron-vervoer ketting komplekse in die mitokondria. Dit lei tot gebrekkige oksidatiewe fosforilering en veroorsaak ‘n toename in die vorming van ROS, wat dan ‘n toename in oksidatiewe stres binne in die sel tot gevolg het. Uiteindelik lei dit dus tot die beskadiging van die mitokondria wat gepaard gaan met fragmentering van die mitokondriese netwerk. Beskadigde mitokondrias word geetiketeer vir afbraking. Hierdie etiketering aktiveer omringende autophagosome wat die beskadigde mitokondrias dan verwyder deur middel van ‘n verswelgende proses genaamd mitophagy. Dit veroorsaak die aktivering van ‘n aantal gekorreleerde sellulêre prosesse wat lei tot apoptose (afsterwing van die sel). Hierdie studie dra by tot die verklaring van die spesifieke effek wat parkin mutasies het op die funksionering van die mitokondria. Resultate hier lê ook die grondslag vir toekomstige studies met die doel tot die ontwikkeling van terapeutiese strategeë en biochemiese merkers wat kan bydrae tot die genesing van beide pasiënte met PS, asook pasiënte met ander neurodegeneratiewe afwykings.

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.
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.

Next-generation sequencing methods now allow rapid and cost-effective sequencing of DNA on a scale not previously possible. This offers great opportunities for the research of Mendelian disorders, but also significant challenges. The sequencing of exomes, or whole genomes, has emerged as a powerful clinical research tool, with targeted gene analyses generally being preferred in the clinical diagnostic setting. These methods have been employed here with the aim of identifying novel genetic causes of inherited heart disorders and to gain insights into the utility and limitations of these techniques for clinical diagnosis in these disorders. Data produced from the introduction of a targeted multi-gene next-generation sequencing test into clinical practice has been studied. Variation within the mitochondrial genome has been analysed to assess the importance of mitochondrial DNA variants in patients with hypertrophic cardiomyopathy. The m.4300A>G mutation is identified as an important cause of this disorder, with other previously cardiomyopathy-associated and novel variants also identified. Such multi-gene tests can facilitate interpretable and phenotype-relevant results, but at the expense of limiting more extensive data acquisition. Whole-genome sequencing has been performed in five families with different autosomal dominant inherited heart disease phenotypes of unknown genetic aetiology. In two of these likely pathogenic variants were identified, one in the gene encoding titin (TTN) and the other in the calcium channel subunit gene CACNA1C. In vitro studies were undertaken to support the pathogenicity of the TTN variant and understand the functional effects of this. In the other three families either multiple candidate gene variants were identified or no clear candidate variant was identified. This highlights the difficulties in interpreting these results, even in carefully selected families. Overall, although the research benefits of exome or genome studies are evident, the interpretation and validation of genetic variant data produced remains highly challenging for clinical diagnosis.

Ankylosing spondylitis (AS) is a common inflammatory arthritis of the spine and other affected joints, which is highly heritable, being strongly influenced by the HLA-B27 status, as well as hundreds of mostly unknown genetic variants of smaller effect. The aim of my research was to confirm some of the previously observed genetic associations and to identify new associations, many of which are in biological pathways relevant to AS pathogenesis, most notably the IL-23/T_H17 axis (IL23R) and antigen presentation (ERAP1 and ERAP2). Studies presented in this thesis include replication and refinement of several potential associations initially identified by earlier GWAS (WTCCC-TASC, 2007 and TASC, 2010). I conducted an extended study of IL23R association with AS and undertook a meta-analysis, confirming the association between AS and IL23R (non-synonymous SNP rs11209026, p=1.5 x 10-9, OR=0.61). An extensive re-sequencing and fine mapping project, including a meta-analysis, to replicate and refine the association of TNFRSF1A with AS was also undertaken; a novel variant in intron 6 was identified and a weak association with a low frequency variant, rs4149584 (p=0.01, OR=1.58), was detected. Somewhat stronger associations were seen with rs4149577 (p=0.002, OR=0.91) and rs4149578 (p=0.015, OR=1.14) in the meta-analysis. Associations at several additional loci had been identified by a more recent GWAS (WTCCC2-TASC, 2011). I used in silico techniques, including imputation using a denser panel of variants from the 1000 Genomes Project, conditional analysis and rare/low frequency variant analysis, to refine these associations. Imputation analysis (1782 cases/5167 controls) revealed novel associations with ERAP2 (rs4869313, p=7.3 x 10-8, OR=0.79) and several additional candidate loci including IL6R, UBE2L3 and 2p16.3. Ten SNPs were then directly typed in an independent sample (1804 cases/1848 controls) to replicate selected associations and to determine the imputation accuracy. I established that imputation using the 1000 Genomes Project pilot data was largely reliable, specifically for common variants (genotype concordence~97%). However, more accurate imputation of low frequency variants may require larger reference populations, like the most recent 1000 Genomes reference panels. The results of my research provide a better understanding of the complex genetics of AS, and help identify future targets for genetic and functional studies.

博士
國立陽明大學
生物化學研究所
93
In the postgenomic era, sequence annotation is the essential and the first step for genome interpretation. Thus, the first part of this thesis is on the seqeunce annotation. The established pipeline was applied to annotate the draft sequence between 4q22 to 4q25 regions on the human genome. I found 254 genes in 104 BAC clones. There were 43 known genes, 27 related genes that were similar to known genes, 6 pseudogenes and 184 predicted genes that were similar to expressed sequence tag sequences. The second part of my thesis is devoted to the medical application of genome annotation. Since the positional candidate gene approach accelerates the discovery of disease gene, I have made a Spinocerebellar ataxia (SCA) candidate gene database. By using the SCA subtypes that have known disease genes as true positives, I have optimized the condition to search for SCA candidate genes in human. The known disease genes were found in the top 3 candidates by using the default parameters. This database is available at http://ymbc.ym.edu.tw/sca/. The third part of this thesis is focusing on the discovery and annotation of novel genes. In order to overcome all the problems of the prediction method based on EST analysis, I have used a genome-wide motif scanning approach. This method is quite sensitive, so it may identify death domain in draft or even pre-draft sequences. In total I have identified 36 death domain containing protein genes, in which 6 novel genes can be identified in the pre-draft sequences. For those fragments that belong to a known cDNA or protein, the known sequence can be used as a template to annotate the gene structure. I have further performed phylogenetic analysis on death domain sequences. The domain analysis alone yielded a similar tree to that of a superfamily analysis. As a result, the paralogous gene in a group may also be used as a template to annotate novel genes in the same group. After annotating the gene structure of these genes, I further observed that genes in the same group usually have identify splicing frames. Thus, I have established a pipeline for phylogenetic analysis of domains(http://ymbc.ym.edu.tw/pad/). Hopefully, this tool will accelerate the discovery of novel genes that contain other protein domains in human, mouse, and fly genomes.

碩士
國立臺灣大學
生醫電子與資訊學研究所
106
Alzheimer''s disease (AD) is a condition that worsens over time, and symptoms can gradually deteriorate from memory loss to loss of mobility and ultimately death. At present, the cause of AD is only speculated. The real cause is still unclear, and the current treatment can only delay the rate of deterioration, but the disease cannot be cured. Although previous genome-wide association studies have identified several risk factors for this disease, these factors can only be used to predict the risk of an individual. The biggest disadvantage of such approaches is that only the effects of a single factor are considered and the effects of gene combinations are ignored. The current research method for the discovery of compound effect, that is, Epistasis, whether based on statistical linear methods or machine learning methods, requires a large memory capacity to detect all possible combinations, and the combination is usually limited to two elements. In this regard, this study aims at replacing current methods for epistasis with multi-layer perceptron, which is one kind of deep learning methods, to predict individual phenotypes from its genotype data, as well as interpreting certain epistasis from our model. The material for this study was derived from 364 individuals in Alzheimer’s Disease Neuroimaging Initiative (ADNI). These individuals were diagnosed as AD or cognitively normal (CN) control. This study established single-gene model and cross-gene model in sequence, and found within-gene and cross-gene epistasis from these two models. The genetic features found included some known important risk factors, such as APOE-ε4. This proves that the deep learning model of this study can indeed find some important combinations in real data. In addition, this research method has also found combinations with more than two elements, which solves the limitation of the current methods.

碩士
國立臺灣師範大學
生命科學研究所
95
Parkinson’s disease (PD) is the second most common neurodegenerative disorder characterized by resting tremor, rigidity, bradykinesia, and postural instability. Besides, the pathological features are the presence of intraneural inclusions, Lewy bodies, and selected loss of neurons in the substantia nigra. Based on previous studies, parkin nad DJ-1gene involved in autosomal recessive juvenile parkinsonism (AR-JP), which characterized by early onset and pathology demonstrated loss of the neurons in substantia nigra but absence of Lewy body. On the other hand, it has been suggested that mitochondrial dysfunction could be involved in PD, and several mitochondrial single-nucleotide polymorphism (SNP) have been reported. Therefore, we detect parkin and DJ-1 mutions in Taiwanese population. Direct sequencing of the parkin and DJ-1 gene found two deletion(Δ138Ala and Δexon5), two reported SNP and seven novel point mutation. We also analyzed whether these three genetic polymorphisms are associated with PD in a cohort of 416 PD cases and 372 ethnically matched controls. The allele frequency distribution of any of these three analyzed polymorphisms was not significantly different between the cases and the controls. None of the six haplotypes derived influences risk of PD. Notably, after stratification by age, individuals over 70 years of age carrying the haplotype 9055G-10398A-13708G demonstrated a significant decrease in risk of developing PD (OR = 0.44, 95% CI = 0.24-0.80, p = 0.008). These results suggest that the mtDNA haplotype 9055G-10398A-13708G plays a role in PD susceptibility among Taiwanese people older than 70 years of age