Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Progressive familial heart block I (PFHBI) is an inherited autosomal dominant cardiac
conduction disorder which segregates in a large South African (SA) pedigree, two
smaller SA families and a Lebanese family. It specifically affects conduction in the
ventricles and is of unknown cause. Clinically, PFHBI is detected on electrocardiogram
(ECG) by evidence of bundle-branch disease, i.e., as right bundle branch block, left
anterior or posterior hemiblock, or complete heart block with broad QRS complexes. The
PFHBI-causative gene was mapped to a lOcM region on chromosome 19ql3.3 using
linkage analysis, and the locus was subsequently reduced to 7cM by genetic fine
mapping.
The present study involved a multi-strategy approach to search for the PFHBI gene. The
objectives were the further reduction of the PFHBI locus by genetic fine mapping using
published and novel markers, searching for short gene transcripts from publicly available
databases and the generation of an integrated map of the locus to which genes were
mapped. Prioritised genes were screened for PFHBI-causing mutations and, in addition,
the PFHBI locus was searched for the presence of a G protein-encoding gene (PI 15-
RhoGEF), a connexin (Cx) gene and any genes containing a CTG repeat expansion motif,
since these genes are plausible PFHBI candidate genes.
Genotyping and fine genetic mapping using known and novel polymorphic dinucleotide
(CA)n and novel tetranucleotide (A3G)n repeat markers across the PFHBI locus were
performed. Publicly available databases, such as LLNL (Livermore, USA), and
GENEMAP (NCBI) were searched for ESTs which, in turn, were extended using
clustering programmes, such as UNIGENE (NCBI) and STACK (SANBI), and the
resulting consensus sequences were subsequently BLAST-searched against the protein
databases. Using the available data, an integrated physical and genetic map of the PFHBI
locus was generated and, as the HGP progressed, a number of novel genes were placed
thereon. Subsequently, genes were prioritised on the basis of position, function and expression profile.
Genetic fine mapping reduced the PFHBI locus from 7cM to 4cM. The EST approach
yielded 38 ESTs, of which 24 ESTs matched proteins, such as activating transcription
factor 5 (ATF5), actin-binding protein (KPTN) and zinc finger protein 473 (ZFP473)
(May 2003). All the map data generated experimentally and computationally were placed
on the PFHBI map. The PI 15-RhoGEF was excluded as a PFHBI candidate gene and
although homologous sequences to connexin 37 (Cx37) was located on both chromosome
19 radiation hybrid clones (RHG12 and ORIM-7), it was not identified on the DNA
clones spanning the PFHBI locus. No evidence of an expansion of a CTG repeat motif
sequence in PFHBI-affected individuals was found. Five highly prioritised candidate
genes, namely, 5CZ2-associated X protein (BAX), potassium voltage-gated channel
Shaker-related subfamily member 7 (KCNA7’), potassium inwardly-rectifying channel,
subfamily J, member 14 (KIR2.4), lin-7 homolog B {LIN-7B) and glycogen synthase 1
(GSYI) were selected for mutation screening. No disease associated mutations were
identified in the exonic and flanking intronic regions of these genes.
In summary, this study reduced the PFHBI locus substantially and generated a detailed
map of the region. A number of attractive candidate genes were excluded from causing
PFHBI; however, several plausible candidate genes are still present at this gene-rich
locus and remain to be screened. Identifying the PFHBI-causative gene and associated
mutation will provide a platform for further studies to understand the pathophysiology,
not only of PFHBI, but also of other more commonly occurring conduction disturbances. / AFRIKAANSE OPSOMMING: Progressiewe familiele hartblok I (PFHBI) is ‘n autosomaal dominant oorerflike kardiale
geleidingstoomis wat in ‘n groot Suid-Afrikaanse (SA) familie, twee kleiner SA families en ‘n
Lebanese familie segregeer. Dit affekteer hoofsaaklik die geleiding in die ventrikels en die oorsaak
daarvan is onbekend. Klinies word PFHBI op elektrokardiogram (EKG) geidentifiseer as a
bondeltak-siekte, naamlik, as regter bondeltakblok, linker anterior of posterior hemiblok, of
volledige hartblok met wye QRS komplekse. Die PFHBI-veroorsakende geen is voorheen deur
koppelingsanalise tot ‘n lOcM gebied op chromosoom 19ql3.3 gekarteer, en daaropvolgens is die
lokus verklein tot 7cM deur genetiese fyn kartering.
Die huidige studie behels ‘n veelvuldige-strategie benadering in die soektog na die PFHBI geen.
Die doel van die studie was die verdere verkleining van die PFHBI lokus deur gebruik te maak van
beide gepubliseerde en nuwe genetiese merkers, die identifisering van kort geentranskripte (ESTs)
uit publieke databanke en die generasie van ‘n geintegreerde kaart van die lokus. Geprioritiseerde
gene is geanaliseer vir die PFHBI-veroorsakende mutasie en, daarby, is die PFHBI lokus deursoek
vir die teenwoordigheid van ‘n G proteien-enkodeeringsgeen (PIJ5-RhoGEF), ‘n konneksien (Kx)
geen en enige gene wat ‘n uitgebreide CTG-herhalingsmotief bevat, aangesien hierdie gene as sterk
PFHBI kandidaatgene geag is.
Genotipering en fynkartering deur die gebruik van bekende asook nuwe polimorfiese dinukleotied-
[(CA)n] en nuwe tertranukleotied- [(A3G)n] herhalingsmerkers wat die PFHBI lokus oorbrug, is
uitgevoer. Publieke databanke, soos LLNL (Livermore, USA), en GENEMAP (NCBI) is ondersoek
vir ESTs wat vervolgens verleng is deur gebruik te maak van groeperende programme soos
UNIGENE (NCBI) en STACK (SANBI) en die gevolglike konsensus volgordes is daama met
behulp van BLAST geanaliseer teen die protei'endatabanke. Die bekomde data is vervolgens gebruik om ‘n geintegreerde fisiese en genetiese kaart van die PFHBI lokus te produseer en, soos
die mens genoomprojek gevorder het, is nuwe gene daarop geplaas. Daarna is gene geprioritiseer
vir mutasie analise gebaseer op posisie, funksie en uitdrukkingsprofiele.
Genetiese fynkartering het die PFHBI lokus van 7cM tot 4cM verklein. Die EST benadering het 38
ESTs gei'dentifiseer, waarvan 24 ESTs proteien gelyke gehad het, bv aktiverende transkripsie faktor
5 (ATF5), aktien-verbindingsprotei'en (KPTN) en sink-vingerproteien 473 (ZFP473) (Mei 2003). A1
die karterings data wat eksperimenteel en rekenaar-gewys gegenereer is, is op die PFHBI kaart
geposisioneer. Die P115-RhoGEF is uitgeskakel as ‘n PFHBI kandidaatgeen en alhoewel ’n
volgorde met homologie aan konneksien37 (Kx37) gevind is op albei chromosoom 19 radiasiehibried
klone (RGH12 and ORIM-7), is dit nie gei'dentifiseer in die DNS klone wat die PFHBI
lokus oorbrug nie. Geen bewyse van uitbreiding van CTG herhalingsmotiewe is gevind in PFHBIaangetasde
persone nie. Vyf hoogs-geprioritiseerde kandidaat gene, naamlik, BCL2-geassosieerde
X proteien (BAX), kalium spanningsbeheerde kanaal, subfamilie J, lid 14 (KIR2.4), lin-7 homoloog
B (LIN-7b) en glikogeen sintase 1 (GYS1), is geselekteer vir mutasie-analise. Geen siekteveroorsakende
mutasie is egter gei'dentifiseer in die eksoniese of die naasliggende introniese
gebiede van hierdie gene nie.
Ter opsomming, hierdie studie het die PFHBI lokus verklein en het ‘n omvattende kaart van die
gebied gegenereer. Verskillende kandidaat gene is uitgesluit as die oorsaak van PFHBI, alhoewel
daar nog heelwat goeie kandidaat gene in hierdie geen-ryke lokus is wat geanaliseer behoort te
word. Die identifiseering van die PFHBI-veroorsakende mutasie sal ‘n platform bied vir verdere
studies om die patofisiologie van nie alleen PFHBI nie, maar ook meer algemene
geleidingstoomisse, te verstaan.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/50187 |
| Date | 12 1900 |
| Creators | Arieff, Zainunisha |
| Contributors | Corfield. V. (Valerie), Stellenbosch University. Faculty of Medicine & Health Sciences. Dept. of Biomedical Sciences. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 368 p. : ill. |
| Rights | Stellenbosch University |
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