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Combined linkage analysis and exome sequencing identifies novel genes for familial goiter / 連鎖解析と全エキソン配列決定による家族性甲状腺腫に関連する新規遺伝子の同定Yan, Junxia 24 March 2014 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18142号 / 医博第3862号 / 新制||医||1002(附属図書館) / 31000 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山田 亮, 教授 小杉 眞司, 教授 松田 文彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
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Genetic causes and risk factors associated with phenotypes occurring in mitochondrial disordersKytövuori, L. (Laura) 15 August 2017 (has links)
Abstract
Finding the genetic causes leading to phenotypes of mitochondrial diseases is challenging because of heterogeneity of the disorders and variety of the underlying biochemical defects. In adults, many of the manifestations of mitochondrial diseases cannot be distinguished from the neurodegenerative processes associated with old age. A single mutation or mutations within the same gene can result in a broad range of disorders. Conversely, clinically similar, monogenic disorders may be caused by genes which are governing entirely different cellular pathways. This study investigated the genetic etiology underlying certain symptoms which are characteristic for mitochondrial syndromes, or mimics of the mitochondrial ones. In the first project, we presented the contribution of genetic variation in the Wolfram Syndrome 1 gene to the risk of diabetes mellitus and sensorineural hearing impairment. We also estimated the frequency of a rare pathogenic variation in WFS1. The second project detected a link between the complex phenotype of age-related hearing impairment and the WFS1 gene. Monogenic forms of ARHI are extremely rare and we succeeded in recognizing one Mendelian form of the trait. The third project confirmed the Mitofusin 2 gene causality in the outlier phenotype of Charcot-Marie-Tooth disease. The fourth project described a Finnish family with two affected siblings with adult-onset ataxia, diabetes mellitus, and hypergonadotropic hypogonadism. The found novel mutation in mtDNA, m.8561C>G, was located in the overlapping region of two mitochondrial genes and resulted in an impaired assembly and dysfunctional energy production of mitochondrial ATP synthase. This thesis expands our knowledge about complex neurological phenotypes and identifies not only some causative genes but also outlier phenotypes, which should be noted in clinical practice. / Tiivistelmä
Perintötekijät mitokondriaalisten ja niiden kaltaisten tautien taustalla ovat vaikeasti tunnistettavissa. Tautien kirjo on valtava, ja niihin johtavat biokemialliset syyt ovat moninaisia. Aikuisten mitokondriotaudit voivat jäädä diagnosoimatta, koska oireet voivat peittyä vanhenemiseen liittyviin neurodegeneratiivisiin prosesseihin. Sama mutaatio tai eri mutaatiot samassa geenissä voivat johtaa kliinisesti täysin erilaisiin ilmiasuihin. Toisaalta, kliinisesti samankaltaiset taudit voivat olla geneettisesti ja solubiologiallisesti kirjavia. Tässä tutkimuksessa selvitetään geneettistä etiologiaa tiettyjen mitokondriaalisille ja niiden kaltaisille taudeille tyypillisten oireiden taustalla. Ensimmäisessä osajulkaisussa tunnistetaan geneettisiä riskivariantteja Wolfram Syndrome 1 -geenissä diabeteksen ja kuulonaleneman taustalta. Lisäksi tutkimuksessa estimoidaan harvinaisen tautia aiheuttavan variaation määrää kyseisessä geenissä. Toinen projekti esittelee suomalaisen perheen, jossa myöhään alkaneen kuulonaleneman, ikäkuulon, geneettinen syy paljastuu WFS1-geenistä, jota ei aiemmin ole liitetty kyseiseen ilmiasuun. Yhden geenin aiheuttamat ikäkuulotapaukset ovat todella harvinaisia, koska ikäkuulo on monimutkainen kokonaisuus, johon ympäristötekijöillä on suuri vaikutus. Kolmas osajulkaisu kuvaa potilastapauksia, joiden ilmiasu on epätyypillinen Charcot-Marie-Toothin neuropatia. Tautigeeni on tunnettu Mitofusin 2, mutta sen aiheuttaman taudinkuvat ovat yleensä vakavampia ja varhain alkaneita. Viimeinen osajulkaisu kuvaa suomalaisen perheen, jonka kahden oireisen sisaruksen taustalta löytyy mitokondriaalisen DNA:n uusi mutaatio, joka sijaitsee kahden geenin alueella muuttaen niiden molempien lopputuotetta. Mutaation, m.8561C>G, osoitetaan vaikuttavan mitokondriaalisen ATP-syntaasin rakentumiseen ja energiatuotantoon. Tämä väitöskirja laajentaa geneettistä tietoisuutta neurologisten tautien taustalla ja esittelee uusia geneettisiä syitä ja ilmiasuja, jotka tulisi huomioida kliinisessä työssä terveydenhuollossa.
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Pathophysiology and gene therapy of the optic neuropathy in Wolfram Syndrome / Physiopathologie et thérapie génique de la neuropathie optique associée au Syndrome de WolframJagodzinska, Jolanta 22 December 2016 (has links)
Le Syndrome de Wolfram (SW; OMIM #222300, prévalence 1-9 / 1000 000) est une maladie neurodégénérative, qui se présente avec un début juvénile, intégrant le diabète insipide, diabète sucré, l’atrophie optique (AO), et la surdité. AO est généralement son premier symptôme neurologique, commençant à l’âge de 11 ans et se terminant par la cécité 8 ans plus tard. Malheureusement, un modèle murin du SW approprié aux symptômes ophtalmologiques n'a pas encore été trouvé, donc la recherche de la thérapie pour sauver la vision en est à ces débuts. Dans cette thèse j’ai étudié l’atteinte visuelle de deux modèles de souris mutantes pour le SW et succès d’une approche de thérapie génique (TG) avec le gène humain WFS1.Premièrement, les souris Wfs1exon8del sont été examinées à 3 et 6 mois pour l’acuité visuelle (AV) et la sensibilité aux contrastes (SC) via changements dans le reflexe optomoteur (ROM), la fonction rétinienne neurale par électrorétinogramme (ERG), ainsi que la physiologie de l’œil par la fondoscopie et tomographie par cohérence optique (TCO). De plus, la proportion des cellules ganglionnaire de la rétine (CGRs) et la perte axonale dans le nerf optique (NO) à 7 mois ont été examinés avec marquage anti-Brn3a et microscopie électronique, respectivement. Il y avait une perte progressive de l’AV et la SC chez les souris KO à partir du 1 mois. Elle était accompagnée d'une pâleur du disque optique (DO), d'amincissement de la rétine ainsi que des lésions axonales. Par contre, il n’avait pas de perte des CGRs ni stress du réticulum endoplasmique dans la rétine. Brièvement, les souris KO présentent un phénotype ophtalmique du SW significatif et peuvent servir comme modèle.Deuxièmement, à la recherche d'un autre modèle du SW, les fonctions visuelles de la lignée Wfs1E864K de la souris ont été étudiées. Déjà à 1 mois, les souris Wfs1E864K/E864K avait une perte drastique de la fonction des CGRs, mais en gardant le nombre de cellules à un niveau normal. Ceci a été accompagné par un amincissement de la rétine et d’un sévère dommage du NO, comme montré par le TCO et la fondoscopie, respectivement. En conséquence, les souris Wfs1E864K/E864K, avec leur fort phénotype ophtalmique, pourraient servir comme modèle du SW classique.Enfin, pour enquêter sur les futures options de traitement contre le SW, les souris de la lignée Wfs1exon8del à 1 mois ont subi une TG intravitréenne avec AAV-2/2-CMV-WFS1. Les examens à 3 et 6 mois ont montré une amélioration de l’AV, ainsi que le sauvetage de la pâleur du DO et réduction des lésions axonales chez les souris KO. En outre, aucun effet indésirable lié à des injections TG n’ont été noté. Suivant cette idée, les souris Wfs1E864K/E864K ont également été soumis à la TG intravitréenne, délivrée à P14, mais sans succès.En conclusion, la lignée Wfs1exon8del de la souris est un modèle fiable du SW, y compris les aspects visuels. Je propose le modèle Wfs1E864K/E864K comme une alternative, en particulier pour enquêter sur la fonction de Wfs1 dans l'œil. Enfin, la GT intravitréenne avec WFS1 a un potentiel pour sauver partiellement le phénotype ophtalmique, ouvrant la voie vers le traitement pour les patients du SW / Wolfram Syndrome (WS; OMIM #222300, prevalence 1-9 / 1 000 000) has a juvenile onset and incorporates diabetes insipidus, diabetes mellitus, optic atrophy (OA), and deafness; leading to death in middle age. OA is its first neurological symptom, starting in adolescence and ending with blindness within 8 years. Unfortunately, a suitable WS mouse model comprising ophthalmologic symptoms has not yet been found, therefore the search for its treatment is delayed. In this thesis, I studied visual impairment in two WS mouse models along with a success of a gene therapy (GT) approach with the human WFS1 gene.Firstly, 3 and 6 months old Wfs1exon8del mice were examined for the visual acuity (VA) and contrast sensitivity via changes in the opto-motor reflex (OMR), the neural retinal function via electroretinogram (ERG), as well as the eye physiology via fundoscopy and optic coher-ence tomography (OCT). Also, the proportion of retinal ganglion cells (RGC) and the axonal loss at the age of 7 months were determined with anti-Brn3a immuno-labeling of retinal sections and electron microscopy of optic nerve (ON) sections, respectively. There was a progressive loss of VA and contrast sensitivity in Wfs1exon8del-/- mice, starting already at 1 month of age. It was accompanied by optic disc pallor, retinal thinning as well as axonal damage. However, there was no RGC loss and the endoplasmic reticulum (ER) stress in the retina was at a normal level. It suggested a presence of another cause for the reported degeneration in KO mice; in opposition to what was proposed in the literature. I brief, KO mice exhibit significant WS ophthalmic phenotype.Secondly, in search for another model, visual functions of Wfs1E864K mouse line were investigated. This line was originally a model of Wolfram-like Syndrome, characterized by dominant mutations in WFS1 leading to congenital progressive hearing impairment, diabetes mellitus and OA. Only homozygous mutants, however, showed expected visual impairment. Already at 1 month of age, Wfs1E864K/E864K mice had drastic loss of RGC function, albeit keeping the cell number at a normal level. This was accompanied by retinal thinning and a severe ON damage, as shown with OCT and fundoscopy, respectively. In contrast, the RGC function in Wfs1E864K/+ mice dropped slightly only at the age of 7 and 12 months, showing that the pathology of the E864K mutation-driven disease in mice is different than in humans. Therefore, Wfs1E864K/E864K mice, with their strong ophthalmic phenotype, could potentially serve as a model of the classical WS.Finally, to investigate future treatment options, 1 month old Wfs1exon8del+/+ (WT) and Wfs1exon8del-/- (KO) mice underwent a uni- and bi-lateral intravitreal gene therapy (GT) with AAV-2/2-CMV-WFS1. Exams at 3 and 6 months of age showed improved VA, as well as optic pallor and axonal damage rescue in KO mice. Also, no adverse effects related to either GT or sham injections were noted. Following this idea, the Wfs1E864K/E864K mice were also subjected to intravitreal GT, delivered at P14, but without success.In conclusion, Wfs1exon8del mouse line is a reliable model of WS, including the visual aspects. I propose the Wfs1E864K/E864K model as an alternative, especially to investigate Wfs1 function in the eye. Finally, the intravitreal AAV-driven GT with WFS1 has a potential to partially rescue the ophthalmic phenotype, paving the wave towards the treatment for WS patients.
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Identification of ligands interacting with the Wolframin protein (WFS1), a candidate in the pathophysiology of posttraumatic stress disorder (PTSD)Honing, Candice 03 1900 (has links)
Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Posttraumatic stress disorder (PTSD) is a multifactorial disorder, with substantial evidence for a genetic
contribution. Although genetic association studies have been conducted to identify vulnerability factors
in PTSD, the results remain largely inconsistent. Identifying ligands of proteins that are involved in the
aetiology of PTSD represents a means of delineating the network of interactions that may play a role in
the development of the disorder. Numerous animal studies have identified the Wolframin protein
(WFS1) as a putative biomarker for the development of PTSD. However, the function of WFS1 has not
yet been fully elucidated. The aim of the present investigation was to identify proteins that interact with
the N-terminal domain of WFS1, in order to possibly elucidate the function of the protein, and to
subsequently hypothesise on the role that WFS1 may play in the development of PTSD.
Yeast two-hybrid (Y2H) methodology was used to identify putative ligands of the N-terminal domain
of WFS1 (amino acids 1-300) by screening a human adult brain complementary DNA (cDNA) library.
Successive selection stages reduced the number of putative WFS1 N-terminal ligand-containing
colonies (preys) from 878 to three. Putative ligands were sequenced and indentified by BLAST-search.
Four preys were excluded because they were either out of frame with the vector or the protein they
encoded occurred in a subcellular location that was not compatible with the location of the N-terminal
domain of WFS1. An interesting putative ligand was identified as carboxypeptidase E (CPE).
Colocalisation analyses verified that CPE colocalises with WFS1 in rat hypothalamic GT1-7 cells. Coimmunoprecipitation
(Co-IP) further verified a direct interaction between WFS1 and CPE in rat
hypothalamic GT1-7 cells, providing conclusive evidence that WFS1 and CPE interact.
Both WFS1 and CPE are upregulated in response to fear and both are localised to the secretory
granules of the regulated secretory pathway. WFS1 has been detected in both the ER and secretory
granules it seems to play an important role in protein biosynthesis, modification, folding, trafficking
and the regulation of calcium homeostasis. CPE is involved in neuropeptide processing and trafficking
of secreted proteins. The interaction between CPE and WFS1 may thus serve to facilitate an optimal
environment in which neuropeptides can be processed and secreted. / AFRIKAANSE OPSOMMING: Posttraumatiese stresversteuring (PTSV) is 'n multifaktoriese siekte, met aansienlike bewyse vir 'n
genetiese bydrae. Hoewel genetiese assosiasie-studies uitgevoer word om kwesbaarheidsfaktore in
PTSV te identifiseer, is die resultate grootliks teenstrydig. Identifiseering van ligande van proteїene wat
betrokke is in die etiologie van PTSV dien as middel om die netwerk van interaksies wat ń moontlike
rol in die ontwikkeling van die versteuring kan speel, te oudersoek talle diere studies het die Wolframin
proteien (WFS1) geїdentifiseer as 'n moontlike biomerker vir die ontwikkeling van PTSV. Die funksie
van WFS1 is egter nog nie ten volle beskryf nie. Die doel van die huidige studie was om proteїene wat
interaksie met die N-terminale domein van WFS1 her te identifiseer, om sodoende die funksie van die
proteїen uit te lig, en daardeur die rol wat WFS1 kan speel in die ontwikkeling van PTSV te bepaal.
Die gis twee-hibried metodologie is gebruik om moontlike ligande van die N-terminale domein van
WFS1 te identifiseer, deur die sifting van 'n mens volwasse brein komplementêre DNS
biblioteek. Opeenvolgende seleksie stappe het die aantal moontlike WFS1 N-terminale ligand wat
moontlike prooi kolonies bevat van 878 tot en met ses verminder. Die DNS volgorde van die moontlike
prooi-plasmiede is bepaal en geїdentifiseer deur die BLAST soek-engin. Vier prooi-plasmiede is
uitgesluit omdat hulle of nie in die korrekte lees-raam in die vektor was nie of die subsellulêre ligging
van die proteїen wat uitgedrukword is nie versoenbaar met die N-terminale domein van WFS1. 'n
Interessante moontlike ligand is geїdentifiseer as Karboxypeptidase E (CPE). Ko-lokalisering ontleding
bevestig dat CPE ko-lokaliseer met WFS1 in rot hipotalamiese selle (GT1-7). Ko-immunopresipitasie
(Ko-IP) toon verder 'n direkte interaksie tussen WFS1 and CPE in rot GT1-7 selle. Wat dus bewys dat
WFS1 en CPE wel met mekaar 'n interaksie het.
Beide WFS1 en CPE toon 'n verhoogde uitdrukking in respons tot ń vrees-situasie. Beide van hierdie
proteїene kom voor in die sekretoriese korrels van die gereguleerde sekretoriese pad. Die WFS1
proteien word bevind in die endoplasmiese retikulum (ER) van die sel, waar dit verantwoordelik is vir
proteien biosintese, modifikasie, vouing, vervoer en die reguleering van kalsium homeostase. Die CPE
proteїen is verantwoordelik vir die proseseering van neuropeptiede en die vervoer van uitgeskiede
proteїene. Dus kan die interaksie tussen CPE en WFS1 dien om 'n optimale omgewing te skep waarin
neuropeptiede geproseseer en uitgeskei kan word. / The National Research Foundation (NRF), the Harry Crossley Foundation and the Medical Research Council
(MRC)
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