Thesis (PhD)--University of Stellenbosch, 2004. / 426 Leaves printed single pages, preliminary pages i-xxiv and i-xxvii and 399 numberd pages. Includes bibliography. List of figures, List of tables, List of abbreviations. / ENGLISH ABSTRACT: Hypertrophic cardiomyopathy (HCM) is an autosomal dominantly inherited primary cardiac disease.
The primary features of HCM are left ventricular hypertrophy, myocardial disarray, fibrosis and an
increased risk of sudden cardiac death. To date, more than 264 HCM-causing mutations, occurring in
thirteen genes, have been identified. As the vast majority of HCM-causing mutations occur in
components of the cardiac sarcomere, HCM has been considered a disease of the cardiac sarcomere.
Functional analyses of HCM-causing mutations in sarcomeric protein-encoding genes revealed that
HCM-causing mutations have a vast array of effects on contractile function. The discovery of HCMcausing
mutations in the gamma two subunit of adenosine monophosphate activated protein kinase
highlighted the fact that mutations in non-sarcomeric proteins can also cause HCM and supports a
hypothesis that HCM-causing mutations may result in energy wastage leading to energy depletion.
Mutations in the cardiac myosin binding protein C (cMyBPC) gene (MYBPC3) are the second most
prevalent cause of HCM. cMyBPC is a modular protein that forms an integral part of the sarcomeric
thick filament, where it acts as a regulator of thick filament structure and cardiac contractility.
Although cMyBPC has been studied extensively, the mechanisms through which it fulfill these
functions have remained elusive, largely due to a lack of a comprehensive understanding of its
interactions with other sarcomeric components and its quaternary structure.
The aims of the present study were, firstly, to screen MYBPC3 for HCM-causing mutations in a
panel of HCM-affected individuals and, secondly, to identify the ligands of domains of cMyBPC in
which HCM-causing mutations were found.A panel of deoxyribonucleic acid (DNA) samples obtained from unrelated HCM-affected individuals
was screened for HCM-causing mutations in MYBPC3, using polymerase chain reaction (PCR)-
based single-strand conformation polymorphism method, as well as restriction enzyme digestion,
DNA sequencing and reverse transcription PCR techniques. In order to identify the ligands of
domains in which HCM-causing mutations were found, yeast two-hybrid (Y2H) candidate-ligandand
library-assays were performed.
Three novel and two previously described putative HCM-causing mutations were identified in
MYBPC3. Data generated in this and other studies, however, suggest that two of these “mutations”
are likely to be either polymorphisms, or disease-modifying factors, rather than main-locus HCMcausing
mutations.
Recent findings showed a specific interaction between domains C5 and C8 of cMyBPC. This finding
identified domains C6 or C10 as candidate ligands of domain C7. Y2H-assays revealed a specific
C7:C10 interaction. Additional Y2H assays also identified C-zone titin as a ligand of domain C7 and
domain C10 as a ligand of domain C3. Several other Y2H assays, however, yielded no known
sarcomeric ligands of the N-terminal region of cMyBPC.
Identification of the ligands of specific domains of cMyBPC led to the development of detailed
models of cMyBPC quaternary structure when cMyBPC is both unphosphorylated and fully
phosphorylated. The integration of these models into an existing model of thick filament quaternary
structure allows new insights into the functioning of cMyBPC as a regulator of both thick filament
structure and cardiac contractility, as well as the pathophysiology of cMyBPC-associated HCM. / AFRIKAANSE OPSOMMING: Hipertrofiese kardiomiopatie (HKM) is ‘n outsosomaal dominante primêre hartsiekte. Die primêre
kenmerke van HKM is linker ventrikulêre hipertrofie, miokardiale wanorde, fibrose en ‘n verhoogde
risiko van skielike dood. Tot dusver is 260 HKM-veroorsakende mutasies in 13 gene geïdentifiseer.
Aangesien die oorgrote meerderheid van HKM-veroorsakende mutasies in komponente van die
kardiale sarkomeer voorkom, is HKM as ‘n siekte van die kardiale sarkomeer beskryf. Funksionele
analise van HKM-veroorsakende mutasies in sarkomeriese protein-koderende gene het aan die lig
gebring dat hierdie mutasies ‘n wye spektrum van gevolge op kontraktiele funksie het. Die
ontdekking van HKM-veroorsakende mutasies in die gamma-twee subeenheid van adenosien
monofosfaat-geaktiveerde proteïen kinase het die feit dat mutasies nie-sarkomeriese proteïene ook
HKM kan veroorsaak onderstreep en ondersteun ‘n hipotese dat HKM-veroorsakende mutasies
energievermorsing en energie uitputting tot gevolg het.
Mutasies in die kardiale miosien-bindingsproteïen C (kMiBPC) geen (MYBPC3) is die tweede mees
algemene oorsaak van HKM. kMiBPC is ‘n modulêre protein wat ‘n integrale deel van die
sarkomeriese dik filament vorm, waar dit die struktuur van die dik filament en kardiale kontraktiliteit
reguleer. Nieteenstaande die feit dat kMiBPC intensief bestudeer is, word die meganismes hoe
hierdie funksies vervul word swak verstaan, grotendeels weens die afwesigheid van ‘n in diepte
begrip van sy interaksies met ander komponente van die sarkomeer asook sy kwaternêre struktuur.
Die doelstellings van hierdie studie was, eerstens, om MYBPC3 vir HKM-veroorsakende mutasies in
‘n paneel van HKM-geaffekteerde individue te deursoek en tweedens, om die ligande van domeine
van kMiBPC waarin HKM-veroorsakende mutasies gevind is te identifiseer.‘n Paneel van deoksiribonukleïensuur (DNS) monsters verkry van onverwante HKM-geaffekteerde
individue is deursoek vir HKM-veroorsakende mutasies in MYBPC3, deur middel van die polimerase
ketting-reaksie (PKR)-gebasseerde enkelstrand konformasie polimorfisme metode, sowel as
restriksie ensiem vertering, DNS volgordebepaling en terugtranskripsie PKR tegnieke. Die ligande
van domeine van kMiBPC waarin HKM-veroorsakende mutasies gevind is, is geïdentifiseer deur
middel van gis twee-hibried (G2H) kandidaat-ligand en biblioteek-siftings eksperimente.
Drie onbeskryfde en twee voorheen beskryfde vermeende HKM-veroorsakende mutasies in
MYPBC3 is geïdentifiseer. Data gegenereer in hierdie en ander studies dui daarop dat twee van
hierdie “mutasies” eerder polimorfismes, of siekte-modifiserende faktore, as hoof-lokus HKMveroorsakende
mutasies is.
Onlangse bevindings het ‘n spesifieke interaksie tussend die C5 en C8 domeine van kMiBPC getoon.
Hierdie bevindings het óf domein C6, óf C10, as kandidaat-ligande van domein C7 geïdentifiseer.
G2H eksperimente het ‘n spesifieke interaksie tussen domains C7 en C10 getoon. Addisionele G2H
eksperimente het ook C-zone titin as ‘n ligand van domein C7 sowel as domein C10 as ‘n ligand van
domein C3 geïdentifiseer. Verdere G2H eksperimente het egter geen sarkomeriese ligande van die
N-terminale gedeelte van kMiBPC geïdentifiseer nie.
Die identifikasie van ligande van spesifieke domeins van kMiBPC het gelei tot die ontwikkelling van
‘n gedetaileerde model van kMiBPC kwaternêre struktuur wanneer kMiBPC beide ongefosforileerd
en ten volle gefosforileerd is. Die intergrasie van hierdie modelle in bestaande modelle van dik
filament kwaternêre struktuur werp nuwe lig op die funksionering van kMiBPC as ‘n reguleerder van
beide dik filament struktuur en kardiale kontraktiliteit, sowel as die patofisiologie van kMiBPCgeassosieerde
HKM.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/16032 |
| Date | 12 1900 |
| Creators | De Lange, W. J. (Willem Jacobus) |
| Contributors | Corfield, Valerie A., Moolman-Smook, Johanna C., University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xxiv, 398 leaves : ill. |
| Rights | University of Stellenbosch |
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