1) Functional analysis of the polycystic kidney disease proteins polycystin-1 and polycystin-2.

Department                                  :  Dept. of Human and Clinical Genetics

Project supervisor                      :  Dr. D.J.M. Peters/ Drs Hang Le

Address                                       :  Sylvius Laboratories, Wassenaarseweg 72, 2333 AL Leiden

Telephone number                     :  071-5276048

Fax number                                  :  071-5276075

E-mail address                             :  d.j.m.peters@lumc.nl  N.H.Le@lumc.nl

Functional analysis of the polycystic kidney disease proteins polycystin-1 and polycystin-2.

Background of research project      :  Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited disorders.  The disease is characterized by the formation of fluid-filled cysts in both kidneys, leading to chronic renal failure. Two genes have been identified to be mutated in patients with ADPKD, the PKD1-gene, encoding the polycystin-1 protein, and the PKD2-gene, encoding polycystin-2. Our research project is aimed at gaining insight in the function of polycystin-1 and polycystin-2 and in the cellular events which trigger cyst formation as a result of a defect in either or both proteins.

Research questions                      :  Previous work by our and other labs implicate both proteins in cell adhesion, signalling cascades, and cell cycle regulation. To determine critical domains for these functions, mutation deletion constructs are generated and transfected in cells. Luciferase reporter assays, western blotting, and immunofluorescence techniques are performed. To expand our antibody array single chain antibodies are generated from a lama phage bank. Lama derived antibodies are characterized by their high stability and affinity binding to the antigen compared to conventional antibodies. Furthermore, cell lines originating from cystic epithelium of ADPKD patients are assayed for cellular and biochemical differences compared to control cell lines. Within our research field we provide students the opportunity to perform an independent research project, while learning a variety of techniques.

Clinical/Non-clinical                       :  Non-clinical

Methods                                                    :  - DNA techniques: restriction enzyme analysis, DNA isolation, cloning, sequencing (automated), gel-electrophoresis, polymerase chain reaction (PCR), reverse transcriptase-PCR, Northernblot                                                                                        - protein techniques: Western blot, immunoprecipitation, sub-cellular fractionation, lama antibody generation and purification                               

                                                                         - cell culture: cell lines (MDCK, HEK293, HUVEC, ADPKD), primairy cell culture, generation of mutant and knock-out cell lines, transfection, luciferase reporter assay (TOP/FOP)                                                                                     

                                                                         - imaging techniques: immuno-fluorescence, confocal laser scanning and immuno-electron microscopy, enhanced green fluorescent protein fusion constructs for direct and real time fluorescence detection

 

2) Investigations on the regulation of the polycystic kidney disease 1 (PKD1) gene

Department                                  :  Dept. of Human and Clinical Genetics

Project supervisor                      :  Dr. I.S. Lantinga / Dr. D.J.M. Peters

Address                                       :  Sylvius Laboratories

                                                         Wassenaarseweg 72

                                                         2333 AL Leiden

Telephone number                      :  071-5276063/6048

Fax number                                   :  071-5276075

E-mail address                             :  i.s.lantinga@lumc.nl, d.j.m.peters@lumc.nl

 

Background of research project      :     Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most frequent inherited renal disease, with a prevalence of 1:1000. Characteristic for the disease are the formation of fluid-filled cysts in the kidney, effecting renal function and eventually leading to renal failure. In addition, extrarenal manifestations like high blood pressure, cardiac valvular abnormalities, and cysts in liver and pancreas may occur. In 85% of the patients the disease is caused by a mutation in the PKD1-gene. This gene encodes a large protein, polycystin-1, that appears to be involved in cell-cell contacts and signal transduction. To date, little is known about the regulation of the expression of the gene. More insight in the disease may improve therapeutic intervention.

Research questions                :    To gain more insight into the regulation of PKD1-gene expression we aim to analyze the 5'-flanking region of this gene using two approaches: 1) Comparison of PKD1-gene promoter sequences of different species in order to identify (conserved) binding sites for transcription factors. For this purpose, putative PKD1-promoter regions of the mouse and dog will be cloned and sequenced and compared with the human sequence. 2) Using so called 'reporter-constructs', in which different fragments of the  PKD1-promoter region are cloned in front of a reporter-gene (luciferase). We will analyze in vitro which parts of the promoter-region regulate the reporter-gene expression, and thereby probably also the transcription of the PKD1-gene.

Methods                                   :     - Molecular biological: DNA-isolation, restriction enzyme analysis, cloning, sequencing (automatic), gel-electrophoresis, Southern blotting, probe hybridization, polymerase chain reaction (PCR)
- Cell culturing, transfection of cell lines, reporter-gene assay (luciferase)

 

3) Mutation analysis for the Autosomal Dominant Polycystic Kidney Disease 1 (PKD1)-gene

Department                                  :  Dept. of Human and Clinical Genetics

Project supervisor                      :  Dr. D.J.M. Peters

Address                                       :  Sylvius Laboratories, Wassenaarseweg 72, 2333 AL Leiden

Telephone number                     :  071-5276048

Fax number                                  :  071-5276075

E-mail address                             :  d.j.m.peters@lumc.nl

Background of research project  :

Autosomal Dominant polycystic Kidney Disease (ADPKD) is characterised by the formation of fluid-filled cysts in the kidneys and extrarenal manisfestations. The incidence is 1 in 1000.

In 85% of the patients the disease is caused by a mutation in the PKD1-gene located on chromosome 16. This gene encodes a transcript of 14 kb which is for 1/3 unique and for 2/3 highly homologues to a cluster of pseudogenes located on the same chromosome. Sofar, mutation detection has mainly been focussed on the unique part of the gene.

 

Research questions                     :

We have collected DNA of almost 300 families with ADPKD. For a subset of families we want to establish whether the disease is linked to the  PKD1-gene on chromosome 16 or the PKD2-gene on chromosome 4.  The DNA of PKD1-patients will be used to search for mutations.  To search for >small= mutations we will screen exon-by-exon, the >repeated= part of the PKD1-gene.  To analyse the gene for large deletions we want to digest the DNA with restriction enzymes followed by Pulse Field Gel (PFG) Electroforesis and Southern Blotting.

 

Project description and experimental design                             :

The DNA of several recently obtained families will be analysed with polymorfic CA-repeat markers close to the PKD1 gene, for linkage with chromosome 16. When needed, markers near the PKD2 gene on chromosome 4 will be used.

For mutation analysis fragments generated via the  polymerase chain reaction (PCR) will be analysed by electrophoresis on agarose-gels and on polyacrylamide gels (Denaturing gradient gel electrophoresis, Single Stranded Conformation Polymorphism analysis). Mutations will be analysed using an automatic sequencer. To search for large deletions, DNA will be digested with the restriction enzymes and fragments will be separated via gel electrophoresis, followed by Southern blotting and hybridisation with PKD1-probes.

The different techniques will performed in this order but with overlap in time.

 

Techniques to be applied            :

Polymerase Chain Reaction (PCR), CA-repeat analysis (haplotyping),agarose gel electrophoresis, digestion of human DNA, Southern blotting, hybridisation, Single stranded conformation polymorphism analyse (SSCP), Denaturing gradient gel electrophoresis (DGGE). Sequence analysis.