Identifizierung und Charakterisierung neuer Interaktionspartner von Fibrocystin, dem Protein der autosomal rezessiven polyzystischen Nierenerkrankung (ARPKD)

Autosomal recessive polycystic kidney disease (ARPKD) is a monogenetic hereditary disease during the childhood with an incidence of 1:20.000. It is caused by mutations in the gene PKHD1. Phenotypically it is characterized in mainly small-cystic kidneys, congenital hepatic fibrosis, portal hypertension and respiratory insufficiency. The gene PKHD1 has one of the longest open reading frames in the human genome and consists of 66 exons. The gene-product fibrocystin (FPC) has a size of 4074 amino acids and it is a very big protein in comparison to other proteins of the human organism. It is known that FPC contains a transmembrane domain, a very big N-terminal domain and a very small C-terminal domain. The highest expression of FPC exists in kidney, liver and pancreas. Especially, it is detectable in the cortical collecting duct cells of the kidney. Pathologic variations at the primary cilia have been identified as the aetiological factor of ARPKD. This is the reason why ARPKD is a member of the ciliopathies. In the kidney the primary cilia is working as a mechano-, chemo- and osmosensor. The exact pathomechanism of ARPKD has not yet been identified. The aim of this dissertation was therefore to identify further interaction partners of FPC. Furthermore, a method should be established to obtain primary cells of kidney epithel cells which are affected by ARPKD in order to establish cell lines of these primary cells. In this dissertation RACK1 (Receptor for Activated C Kinase 1) could be identified as an interaction partner of FPC. Because of the seven WD40 repeats (formed a beta-propeller structure) it is involved in many signaling pathways like MAPK-kinase, cell migration pathways and cell proliferation pathways. ADAM12 is another interaction partner of FPC. ADAM12 is a zinc depending membrane anchored metalloprotease. Treating the kidney epithel cells with collagenase B and trypsin has been shown as the most efficient method to gain primary cells from kidney epithel cells. For separation and isolation of the cell suspension (fibroblast and kidney epithel cells) the supported Mini-MACS-System (company Miltenyi) were used. For the specific separation of the two types of cells it is possible to use the surface antigen CD40. It is known that FPC starts a Notch-similar pathway by an ectodomain shedding of its N-terminal part. By this mechanism the N-terminal part is separated and binds to the rest of the FPC again and within it activates an intramembrane proteolysis. As a consequence the C-terminal part is separated and translocated to the nucleus and cytosol. ADAM12 is identified as the probable protease which is responsible for the separation of the N-terminal part from FPC. Probably RACK1 is responsible for the correct transport and position of the two proteins during that process

Cystinuria: an inborn cause of urolithiasis

<p>Abstract</p> <p>Cystinuria (OMIM 220100) is an inborn congenital disorder characterised by a defective cystine metabolism resulting in the formation of cystine stones. Among the heterogeneous group of kidney stone diseases, cystinuria is the only disorder which is exclusively caused by gene mutations. So far, two genes responsible for cystinuria have been identified: <it>SLC3A1</it> (chromosome 2p21) encodes the heavy subunit rBAT of a renal b^0,+ transporter while <it>SLC7A9</it> (chromosome 19q12) encodes its interacting light subunit b^0,+AT. Mutations in <it>SLC3A1</it> are generally associated with an autosomal-recessive mode of inheritance whereas <it>SLC7A9</it> variants result in a broad clinical variability even within the same family. The detection rate for mutations in these genes is larger than 85%, but it is influenced by the ethnic origin of a patient and the pathophysiological significance of the mutations. In addition to isolated cystinuria, patients suffering from the hypotonia-cystinuria syndrome have been reported carrying deletions including at least the <it>SLC3A1</it> and the <it>PREPL</it> genes in 2p21. By extensive molecular screening studies in large cohort of patients a broad spectrum of mutations could be identified, several of these variants were functionally analysed and thereby allowed insights in the pathology of the disease as well as in the renal trafficking of cystine and the dibasic amino acids. In our review we will summarize the current knowledge on the physiological and the genetic basis of cystinuria as an inborn cause of kidney stones, and the application of this knowledge in genetic testing strategies.</p

2p21 Deletions in hypotonia-cystinuria syndrome

The significant role of the SLC3A1 gene in the aetiology of cystinuria is meanwhile well established and more than 130 point mutations have been reported. With the reports on genomic deletions including at least both SLC3A1 and the neighboured PREPL gene the spectrum of cystinuria mutations and of clinical symptoms could recently be enlarged: patients homozygous for these deletions suffer from a general neonatal hypotonia and growth retardation in addition to cystinuria. The hypotonia in these hypotonia-cystinuria (HCS) patients has been attributed to the total loss of the PREPL protein. Here we report on the clinical course and molecular findings in a HCS patient compound heterozygote for a new deletion in 2p21 and a previously reported deletion, both identified by molecular karyotyping. The diagnostic workup in this patient illustrates the need for a careful clinical examination in context with powerful molecular genetic tools in patients with unusual phenotypes. The identification of unique genomic alterations and their interpretation serves as a prerequisite for the individual counselling of patients and their families. In diagnostic strategies to identify the molecular basis of both cystinuria and hypotonia 2p21 deletions should be considered as the molecular basis of the phenotype

Mutations in multiple PKD genes may explain early and severe polycystic kidney disease.

To access publisher full text version of this article. Please click on the hyperlink in Additional Links field.Autosomal dominant polycystic kidney disease (ADPKD) is typically a late-onset disease caused by mutations in PKD1 or PKD2, but about 2% of patients with ADPKD show an early and severe phenotype that can be clinically indistinguishable from autosomal recessive polycystic kidney disease (ARPKD). The high recurrence risk in pedigrees with early and severe PKD strongly suggests a common familial modifying background, but the mechanisms underlying the extensive phenotypic variability observed among affected family members remain unknown. Here, we describe severely affected patients with PKD who carry, in addition to their expected familial germ-line defect, additional mutations in PKD genes, including HNF-1β, which likely aggravate the phenotype. Our findings are consistent with a common pathogenesis and dosage theory for PKD and may propose a general concept for the modification of disease expression in other so-called monogenic disorders

Mutations in Multiple PKD Genes May Explain Early and Severe Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is typically a late-onset disease caused by mutations in PKD1 or PKD2, but about 2% of patients with ADPKD show an early and severe phenotype that can be clinically indistinguishable from autosomal recessive polycystic kidney disease (ARPKD). The high recurrence risk in pedigrees with early and severe PKD strongly suggests a common familial modifying background, but the mechanisms underlying the extensive phenotypic variability observed among affected family members remain unknown. Here, we describe severely affected patients with PKD who carry, in addition to their expected familial germ-line defect, additional mutations in PKD genes, including HNF-1 beta, which likely aggravate the phenotype. Our findings are consistent with a common pathogenesis and dosage theory for PKD and may propose a general concept for the modification of disease expression in other so-called monogenic disorders