Publikationsserver der RWTH Aachen University
Molekulare Charakterisierung des PKHD1-Gens und seines Proteins Polyductin bei autosomal rezessiver polyzystischer Nierenerkrankung
Abstract
dc:descriptionAutosomal recessive polycystic kidney diesease is one of the most common diseases affecting the kidney in childhood. The clinical spectrum is highly variable with ranging from peri- or neonatal demise and survival to adulthood respectively. The ARPKD-gene, PKHD1, is located on chromosome 6p12 and the majority of the ARPKD-cases could be ascribed to mutations in this gene. The longest open reading frame comprises 66 exons and encodes a single-transmembrane-protein, called polyductin/ fibrocystin. A genotype-phenotype-correlation exists, that implicates that no patient carrying two truncating mutations survives the peri-/neonatal period. So at least one missense mutation is indispensable for survival of newborns. Within this doctoral thesis four recently identified truncating mutations in five families leading to a surprising mild phenotype where analysed. It was shown that three of them caused a quantitative or qualitative alteration on transcript level. Additionally a possible effect of nonsense mediated decay (NMD) in ARPKD has been shown for the first time and it seems as if NMD may has been circumvendet in at least one of the analysed clinically milder affected cases. Therefore mechanisms above DNA-level modifying the phenotype of ARPKD were revealed. The other main task of this doctoral thesis was a further characterisation of the protein encoded by PKHD1, polyductin, and possible interaction partners. Antibodies against the intracellular C-terminus were generated and established. In this process it has been shown that polyductin localised to the centrosome in HEK293 cells and that the protein participates in the so called midbody during telophase in mitosis. So an involvement of polyductin in cell division concerning mitotic orientation and/or cytokinesis could be hypothesised. The potential binding partner of polyductin analysed during this work, BBS2, is one of the proteins related to the Bardet-Biedl-Syndrom (BBS). BBS is a multisystemic disorder usually concerning pathological alterations of the kidney among others. Immunofluorescence showed a colocalisation of polyductin and BBS2 during all phases of mitosis as well as the interphase. Additional koimmunoprecipitation experiments using different constructs for both, polyductin and BBS2, affirmed the expected interaction. Thereupon a possible connection between polyductin and the BBSome, a complex of seven BBS-proteins including BBS2, was analyzed. BBS7, another member of the BBSome, was shown to interact with BBS2 and revealed a large homology to this protein. So it was tested wether there was link between Polyductin and BBS7. In immunofluorescence experiments a partial colocalisation within the centrosome during the interphase was observed. ARPKD as well as BBS concern to the group of ciliopathies and the localisation and function of the BBSome is described only for polarised ciliated cells. So the ciliar localisation was checked in polarised mIMCD-3 cells. It turned out that polyductin as well as BBS7 could be detected predominantly at the basis and within the cilium. Continuative CoIP experiments indicated the interaction between polyductin and BBS7. Therefore a direct link between the proteins associated with ARPKD and the Bardet-Biedl-Syndrome could be made for the first time. This allows a deeper insight in the complicated network of ciliary signalling and could help understanding and analysing the pathological changes accompanying these and other ciliopathies.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2009
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Frank, Valeska
- Contributors dc:contributor
-
- Zerres, Klaus
Subjects
dc:subject × 12Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- ger